CN103937877B - ALK inhibitor is had first resistance or the identification of rear resistant cancer, it is judged that and treatment - Google Patents

ALK inhibitor is had first resistance or the identification of rear resistant cancer, it is judged that and treatment Download PDF

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CN103937877B
CN103937877B CN201410061691.6A CN201410061691A CN103937877B CN 103937877 B CN103937877 B CN 103937877B CN 201410061691 A CN201410061691 A CN 201410061691A CN 103937877 B CN103937877 B CN 103937877B
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alk
sudden change
labelling
polypeptide
sequence
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CN103937877A (en
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马诺·真田广之
崔英李
苏达学
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Jichi Medical University
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    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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    • G01N2333/91205—Phosphotransferases in general
    • G01N2333/9121—Phosphotransferases in general with an alcohol group as acceptor (2.7.1), e.g. general tyrosine, serine or threonine kinases

Abstract

The invention discloses whether those cancers that can determine that object is suffered from are mutated into the positive to ALK, and/or whether the patient suffering from this cancer has the compound of a relatively slow disease progression process, test kit and method, described ALK sudden change may produce reaction after a kind of ALK inhibitor for treating.The invention also discloses those and can predict suffer from the process that its state of an illness of patient of this cancer develops in time.

Description

ALK inhibitor is had first resistance or the identification of rear resistant cancer, it is judged that and treatment
This divisional application is invention entitled " to have first resistance or the identification of rear resistant cancer to ALK inhibitor, it is judged that and control Treat ", Application No. 2011800058134, the divisional application of the original invention patent application in filing date on February 4th, 2011.
Related application
Application serial filed in the application claim 2010 year 02 month 04 day is the preferential of the U.S. Provisional Application of 61/337,465 Power.
Background of invention
Tyrosine kinase is the tyrosine residue phosphorylation that a class is come on catalytic proteins substrate by transfer end adenosine triphosphate Enzyme.In many cases, tyrosine kinase includes cell proliferation, canceration and the signal transduction aspect of cell differentiation at cell function There is pivotal role.
EML4-ALK is a kind of pattern of fusion protein tyrosine kinase, occurs in nonsmall-cell lung cancer (NSCLC) case 5%, It can cause result (Soda, M.et al. (2007) Nature of small inversion of No. 2 the short arm of a chromosome of the mankind 448:561-566;Mano, H. (2008) Cancer Sci.99:2349-2355).EML4 ALK is by each list The dimeric structure body that body EML4 interacts in coiled-coil domain and causes, thus obtain obvious carcinogenic activity.With turning Gene mouse expresses EML4 ALK, particularly pulmonary epithelial cells, shortly generates various after birth on double lungs Adenocarcinoma tubercle, then after the alk tyrosine kinase activity inhibitor giving oral specially good effect, these tubercles promptly disappear from lung Except (Soda, M.et al. (2008) Proc.Natl.Acad.Sci.USA105:19893-19897).These are observed Showing, in NSCLC canceration, EML4 ALK has the generation kinase whose important function of this fusion, and proof ALK further Inhibitor carrys out the feasibility of molecular targeted therapy cancer.Such as, clinical verification, inhibitor PF-02341066, to ALK and MET All there is tyrosine kinase activity, the positive NSCLC of EML4-ALK treated, its intermittent result more satisfactory (Kwak, E.L.et al. (2009) J.Clin.Oncol.27 (suppl): 15s (abstract3509)).But, EML4-ALK Positive tumor the inhibitor of the unknown molecular composition of Endodontic failure is not responded to.
Except PF-02341066, other tyrosine kinase inhibitors (TKIs) have been demonstrated have aobvious to cancer patient's treatment Activity.Imatinib mesylate, a kind of TKI for ABL1 and KIT, such as, the kinase whose sun of BCR-ABL1 pattern of fusion Property chronic lymphocytic leukemia or KIT positive gastrointestinal stromal tumors case in tool improve significantly effect (Druker, B.J.et al.(2001)N.Engl.J.Med.344:1031-1037;Heinrich,M.C.et al.(2008)J. Clin.Oncol.26:5360-5367).Additionally, gefitinib and Erlotinib, it is all EGF-R ELISA (EGFR) TKIs, treatment activity of EGFR NSCLC in there is effect (Mok, T.S.et al. (2009) J.Clin.Oncol. 27:5080-2087;Mok, T.S.et al. (2009) N.Engl.J.Med.361:947-957).Regrettably, The targeting group of tumor starts just to be difficult to corresponding TKIs merge or just produce drug resistance after beginning to respond to from treatment.Its Secondary, in the case of some failures, also find that cancer target kinases is directly undergone mutation or allosteric affects the shape of ATP-conjunctive tissue, Thus cause combination (Deininger, M.et al. (2005) Blood105:2640-2653 affecting TKI;Kobayashi, S.et al.(2005)N.Engl.J.Med.352:786-792;Pao,W.et al.(2005)PLoS Med.2:e73; Shah, N.P.et al. (2002) Cancer Cell2:117-125).Therefore, in the urgent need to can recognize that at tyrosine The position with mutation on kinases, such as EML4-ALK, in order to preferably development is used for identifying, judging, prevents and treats phase Close the compound of the disease of exception and/or activity expression, test kit and method.
Summary of the invention
The present invention provide at least by identifying the novel anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase that known ALK inhibitor produces resistance (ALK) suddenly change and identify, judge and treat the compound of cancer, method and test kit.Equally, these ALK sudden change can be Clinical characterization pharmaceutical composition, these pharmaceutical compositions can be put into those abnormal ATP-produced by the sudden change of new A LK and combine District, suppresses ALK activity.
On the one hand, the invention provides a kind of for identify one suffer from cancer or have the object suffering from risk of cancer use ALK Whether can produce the method treating unresponsive additional risk during inhibitor for treating, it includes with patient collecting sample and divides Analysis sample checks for the ALK polynucleotide molecule of one or more sudden change, wherein there is the ALK of one or more sudden change Polynucleotide molecule shows that object has had additional risk unresponsive to ALK inhibitor for treating.
Another aspect, the invention provides a kind of for identifying that one suffers from cancer or has the object suffering from risk of cancer and make Whether with producing the method treating unresponsive additional risk during ALK inhibitor for treating, it includes gathering sample with patient This also analyzes sample to check the expression of ALK polypeptide, structure and/or the activity of one or more sudden change, wherein has one Or the ALK polypeptide of multiple sudden change shows that object has had additional risk unresponsive to ALK inhibitor for treating.
In some embodiments of the invention, a kind of ALK inhibitor is not used to treat before object can be, it is also possible to be The most through with a kind of ALK inhibitor for treating, and at least in part to ALK inhibitor (such as PF-02341066, PDD, 2- Methyl isophthalic acid 1-(2-methyl-propyl)-4-oxygen-4,5,6,11,12,13-hexahydro-2H-indazole [5,4-α] pyrroles [3,4-c] Carbazole-8-base [4-(dimethylamino) benzyl] methyl carbamate, (1S, 2S, 3R, 4R)-3-({ 5-chloro-2-[(1-ethyl -2,3,4,5-tetrahydrochysene-6-methoxyl group-2-oxygen-1H-1-benzazepine-7-base) amino]-4-pyrimidine } amino) dicyclo [2.2.1] Hept-5-alkene-2-Methanamide and NVP-TAE684) create resistance.In further embodiments, described cancer includes anaplastic Large celllymphoma, neuroblastoma, breast carcinoma, colorectal cancer, inflammatory myofibroma, nonsmall-cell lung cancer.Also has another In a little embodiments, described sample includes sputum, bronchoalveolar lavage, hydrothorax, organizes, whole blood, serum, blood plasma, Oral cavity is scraped, saliva, cerebrospinal fluid, urine, feces, circulating tumor cell, circle nucleic acid and bone marrow etc..
In also having other embodiments, described sample includes cell or tissue.In certain embodiments, tissue be tumor or Cancerous tissue.In further embodiments, ALK polynucleotide molecule or the polypeptide of one or more sudden changes includes as listed in table 1 Sudden change ALK polynucleotide molecule or polypeptide.In further embodiments, the ALK of one or more sudden changes passes through nucleic acid hybridization Judge.In further embodiments, the ALK of one or more sudden changes is judged by polymerase chain reaction.Real at other Executing in example, one or more ALK polypeptide expression level are with being specifically bound to one or more ALK polypeptide (such as antibody, antibody Derivant, and antibody fragment) reagent find out.In further embodiments, the quantity of one or more ALK polypeptide, Structure and/or activity are compared with controlling sample.In further embodiments, the ALK of one or more sudden changes passes through initial point Time and at least one time put subsequently judge.In further embodiments, sample includes sexual cell or somatic cell base Because of group DNA.
Another aspect, present invention also offers a kind of cancer patient for the treatment of or the method having potential cancer patient, including from trouble Collect sample with person, analyze sample and ascertain whether to there is one or more sudden change ALK polynucleotide molecule as listed in table 1, One is used to treat effective ALK inhibitor then to described patient.In certain embodiments, ALK inhibitor includes
PF-02341066, PDD, 2-methyl isophthalic acid 1-(2-methyl-propyl)-4-oxygen-4,5,6,11,12,13-hexahydro-2H-indazoles [5,4-α] pyrroles [3,4-c] carbazole-8-base [4-(dimethylamino) benzyl] methyl carbamate,
(1S, 2S, 3R, 4R)-3-({ the chloro-2-of 5-[(1-ethyl-2,3,4,5-tetrahydrochysene-6-methoxyl group-2-oxygen-1H-1-benzazepine-7- Base) amino]-4-pyrimidine } amino) dicyclo [2.2.1] hept-5-alkene-2-Methanamide and NVP-TAE684.In other embodiments In, object does not use a kind of ALK inhibitor to treat before can being, it is also possible to through suppressing with a kind of ALK before being Agent is treated, and at least in part ALK inhibitor is created resistance.
Another aspect, present invention also offers a kind of chemosensitivity for determining a cancer patient, so that with a kind of ALK inhibitor carries out the test kit treated, including: a kind of and ALK polynucleotide molecule of one or more sudden changes or polypeptide produce The reagent of raw particular combination；And operation instructions.In certain embodiments, this test kit also includes a kind of ALK inhibitor.? In other embodiments, this reagent includes that one or more polynucleotide probes, each probe include a polynucleotide sequence, This polynucleotide sequence and one of them nucleotide sequence complementary listed by table 1, or with a coding schedule 1 listed by wherein The nucleotide sequence complementary of one polypeptide (such as oligonucleotide, cRNA molecule, RNA molecule and by the synthetic gene of base composition Probe).In further embodiments, probe includes that length is about 50 to 107The polynucleotide of individual nucleotide.Have more also In embodiment, reagent includes a kind of by the one or more nucleotide sequence coded a kind of antibody listed by table 1, and antibody spreads out Biology and antibody fragment are to a peptide species.
Another aspect, present invention also offers a kind of method and determines whether a kind of detection compound controls one or more dashing forward Become the activity of ALK polypeptide, contact including with mammalian cell so that it is transfect a structure, and encode by detection compound The ALK polypeptide of one or more sudden changes, and measure this mammalian cell to determine the work of the ALK polypeptide of one or more sudden change Property, wherein can considerably adjust activity in controlling to express at one due to detected compound, therefore it can be as one or many The actuator of individual sudden change ALK polypeptide.In certain embodiments, the ALK polynucleotide molecule of described one or more sudden changes or Polypeptide includes the sudden change ALK polynucleotide molecule listed by table 1 or polypeptide.In further embodiments, described control includes feeding Breast animal cell expression one wild type ALK polypeptide, polypeptide can be the polypeptide listed by table 1.In further embodiments, one Individual or multiple sudden change ALK polypeptide activity can be that ATP combines, tyrosine kinase activity, cancer cell proliferation, tumor growth, Tumor quantity, apoptosis and neoplasm metastasis.In further embodiments, described control is expressed and is included that mammalian cell exists In the presence of detection compound, express one or more sudden change ALK polypeptide, wherein can be such as with one or more sudden change ALK (such as ATP combines the activity of polypeptide, tyrosine kinase activity, cancer cell proliferation, tumor growth, tumor quantity, cell Apoptosis and neoplasm metastasis) determine the existence of detection compound.
Accompanying drawing explanation
Shown in Fig. 1 is the new A LK sudden change being resistant to alk tyrosine kinase inhibitor of the present invention.Figure 1A is The structural representation of EML4-ALK albumen.Figure shows the position of two de novo sudden changes in kinase domain, and with solid Or hollow arrow respectively illustrates above-mentioned for expanding kinase domain or merging those PCR primer of cDNA.Figure 1B shows The degree of depth sequencing result of ALK kinase domain cDNA.NSCLC cell line H2228 or numbered J-#1, J-#12, J-#113, The PCR primer of the about 1000bp obtained in the sample of J-#127 or LK-#33 GAII system checks order.Kinase domain Total indicator reading (always) and the number of mispairing reading (mispairing) on each position of cDNA represent with blue and Red diamonds respectively. And show in sample J-#1 and J-#113 the insertion (representing with green square) at cDNA5 ' end enlargedly.Fig. 1 C is G4374 And the electrophoretogram of ALK cDNA clone around C4493.PCR cDNA completes, and described cDNA is never to control respectively In the apoplexy due to phlegm (beginning) sampled before treating and the pleural effusion having deteriorated post-sampling, (deterioration) extracts.Substituted A core Thuja acid red display.
Fig. 2 illustrates the gene order of G4374 and C4493 corresponding position in ALK cDNA.In the pleural effusion of sufferer The gene DNA separated can use Platinum@archaeal dna polymerase (Invitrogen, Carlsbad, CA), following primer (5 '-GGTAAGAAGTGGCTCACTCTTGAG-3 ' and 5 '-CACAACAACTGCAGCAAAGACTGG-3 ') carry out PCR, 15s at 94 DEG C, 30s at 60 DEG C, at 68 DEG C 2 minutes, 35 circulations, it is thus achieved that product import to plasmid In pT7Blue-2 (Takara Bio).Then the plasmid 3130xl Genetic Analyser after inserting checks order, to differentiate to contain G4374A (on the left of figure) or C4493A (on the right side of figure) substituted PCR clones.Substituted A nucleotide red display.
Fig. 3 shows the result processing BA/F3 cell with PF-02341066.BA/F3 have expressed EML4-ALK (wild type), EML4-ALK (C1156Y), EML4-ALK (L1196M), or the EML4-ALK (C1156Y/L1196M) of double sudden change, it is in institute Show in the PF-02341066 of concentration and cultivate 48 hours, then observe its cellular morphology with phase contrast microscope.Scale: 20 μm. Fig. 4 shows the performance of the new A LK sudden change being resistant to alk tyrosine kinase inhibitor of the present invention.Fig. 4 A shows 5 ×105Individual BA/F3 cell expresses EML4-ALK (wild type) after cultivating 48h under the shown concentration of PF-02341066, EML4-ALK (C1156Y), the quantity of EML4-ALK (L1196M) or double sudden change EML4-ALK (C1156Y/L1196M).Figure shows Show that BA/F3 expresses the percentage ratio of available cell relative for wild type EML4-ALK.Data refer to three independent experiment obtain ± s.d..Fig. 4 B shows PF-02341066 in the tyrosine phosphorylation effect of wild type or in saltant type EML4-ALK Effect.BA/F3 cell have expressed wild type EML4-ALK or its sudden change of FLAG labelling, is placed on shown concentration In PF-02341066 15 hours, then EML4-ALK immunoprecipitation from cell lysates out, then carried out Diagnosis of Sghistosomiasis Mark analyzes antibody to Tyr1604ALK or the FLAG epi-position (ALK) of phosphorylation. specificity.A kind of EML4-ALK (KM) will be expressed The cell of inactive sudden change is as negative control.Fig. 4 C shows a kind of vitro kinase assessment, and it is thin from BA/F3 that it can be used for assessment The wild type EML4-ALK of immunoprecipitation FLAG labelling out or its sudden change in born of the same parents' (not proceeding in ALK inhibitor).Described Immunoprecipitation be [γ-32P] ATP, the PF-02341066 of a kind of synthetic peptide and described concentration is carried out.Peptide substrates immunity The phosphorylation of precipitation is respectively used to the performance (on the downside of figure) of the anti-FLAG of immunoblotting assay antibody.
Fig. 5 is the three dimensional structure domain model figure of ALK kinase domain.The amino acid position of ALK overlaps Insulin receptor INSR On crystal structure, this Insulin receptor INSR have an ATP congener combined (Japan Protein Information bank in numbered " 1ir3 ", Can inquire about on the WWW of pdbj.org/index.html).Right part of flg shows and obtains on the left of the model shown in left side Protein structure.Α spiral and β-pleated sheet represent by red and yellow respectively.Figure also show spiral α C, Cys1156,and Leu1196Position.
Detailed Description Of The Invention
The present invention at least partial content is to identify specific gene sequence, suddenlys change, so including such as anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase (ALK) Whether rear judgement ALK inhibitor can effectively treat cancer.The most in the present invention, the new A LK gene identified Sudden change (such as the sudden change of EML4-ALK peptide coding) can make polypeptide at least partly ALK inhibitor therapy can be produced resistance.This Bright those use techniques known that also disclose are to the method identifying these specific gene sequence, and these methods are non-limiting Ground includes oligonucleotide gene chip (Brennan, et al. (2004) Cancer Res.64 (14): 4744-8;Lucito,et al.(2003)Genome Res.13:2291-2305;Bignell et al.(2004)Genome Res.14:287-295; Zhao, et al (2004) Cancer Research, 64 (9): 3060-71) and other method of the present invention, such as wrap Include polymerase chain reaction (PCR) and direct sequencing.The invention also discloses the diagnostic kit used in these methods.
Other side of the present invention will be described in further detail below.
I. define
" one " and " a kind of " herein refers to the grammar object of one or more (such as at least one).Such as, " one Kind of composition " a kind of composition or more than one composition.
The copy number that " variable quantity " of terms tag or " change level " of labelling relate to labelling or chromosomal region is increased or decreased, As ALK gene is suddenlyd change and/or gene outcome (labelling as listed in table 1), and/or in a cancer sample, one or one A little expression of specific marker genes with control in sample at one, compared with the expression of marked copies amount, the expression being increased or decreased. " the optional amount " of terms tag is also included within one such as cancer sample equal samples, a labelling and the normal mark controlled in sample Note protein level is compared, the protein level being increased or decreased.
" expression of change " of the sudden change of term ALK gene and/or gene outcome (labelling as listed in table 1) refers to one Individual detection sample, as the sample of collection with a condition subject relates to expression or the copy number of a labelling, they More than or less than expression or the copy number of standard error test, one can be at least and control sample (as being not suffering from cancer from one With the health objects of disease gather sample) in ALK gene sudden change and/or the expression of gene outcome (labelling as listed in table 1) Level or the twice of copy number, Liang Sanbei, two or four times, two or five times or 20 times or more times, or at various control samples In, ALK gene sudden change and/or the Average expression level of gene outcome (labelling as listed in table 1) or copy number.The table of change Reach expression or copy number that level is tested more than or less than standard error, one can be at least and control sample (as from one With the most cancered health objects gather sample) in ALK gene sudden change and/or gene outcome (labelling as listed in table 1) Expression or the twice of copy number, three times, four times, in five times or ten times or more times, or various control sample, ALK gene sudden change and/or the Average expression level of gene outcome (labelling as listed in table 1) or copy number.
" the change activity " of terms tag relates to the activity of a labelling, when sick state, can compare as in a cancer sample The normal labelling activity controlled in sample is higher or lower.The change activity of labelling can be such as to be changed by the expression of labelling, mark Note protein level change, labelling structure change, as with other protein as labelling in identical or different path A reaction changed is occurred to cause.
" the structure change " of terms tag relates to the existence of sudden change, or marker gene or the sudden change of labelled protein, as relatively Normal or wild type gene or protein, affect the sudden change of marker expression or activity.Such as, sudden change includes inserting without limitation Enter and intrinsic stain body weight group, replace, delete and insertion mutation.Sudden change can occur in the coding region of labelling or uncoded district.
In the present invention, " anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase " and " ALK " is same connotation, refers both to any source (as Rodents moves Thing, the mankind and other mammals) primary anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase and it some change and sudden change.In some embodiments In, alk protein matter is expressed by the NCBI of reference number identifier NP_004295.In addition to identifying, this term further relates to people Body protein.In the present invention, described gene code ALK is referred to as " ALK ".In certain embodiments, ALK nucleoside Acid sequence can be 29029631 expression by NCBI and the GenBank accession number of reference number identifier NM_004304.3 sum, Those of ordinary skill in the art can identify that sequence that the present invention is correlated with is (such as coding, 5' end noncoding region, the non-volume of 3' end simply Code district, transcripting starting, translation starts, and transcribes the sequences such as stopping, translation stopping).
The most in the present invention, " anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase " and " ALK " also includes known to a person of ordinary skill in the art ALK fusion protein kinases and various mutant thereof.These ALK fusion protein kinases and various mutant thereof include that alk protein swashs Enzymatic activity, and sudden change of the present invention is translated into the alk protein kinase activity of anti-ALK inhibitor.Typical example includes EML4-ALK suddenlys change 1 (AB274722.1;BAF73611.1), EML4-ALK sudden change 2 (AB275889.1;BAF73612.1), EML4-ALK suddenlys change 3a (AB374361.1;BAG55003.1), EML4-ALK sudden change 3b (AB374362.1;BAG55004.1), EML4-ALK suddenlys change 4 (AB374363.1;BAG75147.1), EML4-ALK sudden change 5a (AB374364.1;BAG75148.1), EML4-ALK suddenlys change 5b (AB374365.1;BAG75149.1), EML4-ALK sudden change 6 (AB462411.1;BAH57335.1), EML4-ALK suddenlys change 7 (AB462412.1;BAH57336.1),KIF5B-ALK(AB462413.1;BAH57337.1), NPM-ALK,TPM3-ALK,TFGXL-ALK,TFGL-ALK,TFGS-ALK,ATIC-ALK,CLTC-ALK,MSN-ALK, TPM4-ALK, MYH9-ALK, RANBP2-ALK, ALO17-ALK and CARS-ALK (for example, see Pulford et al., (2004) J.Cell.Physiol.199:330-358, incorporated by reference of the present invention).Additionally, those of ordinary skill in the art understands one Planting ALK kinases can make the increase of alk protein kinase mutant (as EML4 at least can be with exon with merging especially of its fusant generation 2,6a, 6b, 13,14 and/or 15 merge, such as such as Horn and Pao, (2009) J.Clin.Oncol.27:4247-4253 Described, incorporated by reference of the present invention).Such as, of the present invention typical case ALK sequence is presented herein below:
Table 1
Wild type ALK cDNA sequence (NM_004304.3；GI:29029631):
1gggggcggca gcggtggtag cagctggtac ctcccgccgc ctctgttcgg agggtcgcgg
61ggcaccgagg tgctttccgg ccgccctctg gtcggccacc caaagccgcg ggcgctgatg
121atgggtgagg agggggcggc aagatttcgg gcgcccctgc cctgaacgcc ctcagctgct 181gccgccgggg ccgctccagt gcctgcgaac tctgaggagc cgaggcgccg gtgagagcaa 241ggacgctgca aacttgcgca gcgcgggggc tgggattcac gcccagaagt tcagcaggca 301gacagtccga agccttcccg cagcggagag atagcttgag ggtgcgcaag acggcagcct 361ccgccctcgg ttcccgccca gaccgggcag aagagcttgg aggagccaaa aggaacgcaa 421aaggcggcca ggacagcgtg cagcagctgg gagccgccgt tctcagcctt aaaagttgca 481gagattggag gctgccccga gaggggacag accccagctc cgactgcggg gggcaggaga 541ggacggtacc caactgccac ctcccttcaa ccatagtagt tcctctgtac cgagcgcagc 601gagctacaga cgggggcgcg gcactcggcg cggagagcgg gaggctcaag gtcccagcca 661gtgagcccag tgtgcttgag tgtctctgga ctcgcccctg agcttccagg tctgtttcat 721ttagactcct gctcgcctcc gtgcagttgg gggaaagcaa gagacttgcg cgcacgcaca 781gtcctctgga gatcaggtgg aaggagccgc tgggtaccaa ggactgttca gagcctcttc 841ccatctcggg gagagcgaag ggtgaggctg ggcccggaga gcagtgtaaa cggcctcctc 901cggcgggatg ggagccatcg ggctcctgtg gctcctgccg ctgctgcttt ccacggcagc 961tgtgggctcc gggatgggga ccggccagcg cgcgggctcc ccagctgcgg ggccgccgct 1021gcagccccgg gagccactca gctactcgcg cctgcagagg aagagtctgg cagttgactt 1081cgtggtgccc tcgctcttcc gtgtctacgc ccgggaccta ctgctgccac catcctcctc 1141ggagctgaag gctggcaggc ccgaggcccg cggctcgcta gctctggact gcgccccgct 1201gctcaggttg ctggggccgg cgccgggggt ctcctggacc gccggttcac cagccccggc 1261agaggcccgg acgctgtcca gggtgctgaa gggcggctcc gtgcgcaagc tccggcgtgc 1321caagcagttg gtgctggagc tgggcgagga ggcgatcttg gagggttgcg tcgggccccc 1381cggggaggcg gctgtggggc tgctccagtt caatctcagc gagctgttca gttggtggat 1441tcgccaaggc gaagggcgac tgaggatccg cctgatgccc gagaagaagg cgtcggaagt 1501gggcagagag ggaaggctgt ccgcggcaat tcgcgcctcc cagccccgcc ttctcttcca 1561gatcttcggg actggtcata gctccttgga atcaccaaca aacatgcctt ctccttctcc 1621tgattatttt acatggaatc tcacctggat aatgaaagac tccttccctt tcctgtctca 1681tcgcagccga tatggtctgg agtgcagctt tgacttcccc tgtgagctgg agtattcccc 1741tccactgcat gacctcagga accagagctg gtcctggcgc cgcatcccct ccgaggaggc 1801ctcccagatg gacttgctgg atgggcctgg ggcagagcgt tctaaggaga tgcccagagg 1861ctcctttctc cttctcaaca cctcagctga ctccaagcac accatcctga gtccgtggat 1921gaggagcagc agtgagcact gcacactggc cgtctcggtg cacaggcacc tgcagccctc 1981tggaaggtac attgcccagc tgctgcccca caacgaggct gcaagagaga tcctcctgat 2041gcccactcca gggaagcatg gttggacagt gctccaggga agaatcgggc gtccagacaa 2101cccatttcga gtggccctgg aatacatctc cagtggaaac cgcagcttgt ctgcagtgga 2161cttctttgcc ctgaagaact gcagtgaagg aacatcccca ggctccaaga tggccctgca 2221gagctccttc acttgttgga atgggacagt cctccagctt gggcaggcct gtgacttcca 2281ccaggactgt gcccagggag aagatgagag ccagatgtgc cggaaactgc ctgtgggttt 2341ttactgcaac tttgaagatg gcttctgtgg ctggacccaa ggcacactgt caccccacac 2401tcctcaatgg caggtcagga ccctaaagga tgcccggttc caggaccacc aagaccatgc 2461tctattgctc agtaccactg atgtccccgc ttctgaaagt gctacagtga ccagtgctac 2521gtttcctgca ccgatcaaga gctctccatg tgagctccga atgtcctggc tcattcgtgg 2581agtcttgagg ggaaacgtgt ccttggtgct agtggagaac aaaaccggga aggagcaagg 2641caggatggtc tggcatgtcg ccgcctatga aggcttgagc ctgtggcagt ggatggtgtt 2701gcctctcctc gatgtgtctg acaggttctg gctgcagatg gtcgcatggt ggggacaagg 2761atccagagcc atcgtggctt ttgacaatat ctccatcagc ctggactgct acctcaccat 2821tagcggagag gacaagatcc tgcagaatac agcacccaaa tcaagaaacc tgtttgagag 2881aaacccaaac aaggagctga aacccgggga aaattcacca agacagaccc ccatctttga 2941ccctacagtt cattggctgt tcaccacatg tggggccagc gggccccatg gccccaccca 3001ggcacagtgc aacaacgcct accagaactc caacctgagc gtggaggtgg ggagcgaggg 3061ccccctgaaa ggcatccaga tctggaaggt gccagccacc gacacctaca gcatctcggg 3121ctacggagct gctggcggga aaggcgggaa gaacaccatg atgcggtccc acggcgtgtc 3181tgtgctgggc atcttcaacc tggagaagga tgacatgctg tacatcctgg ttgggcagca 3241gggagaggac gcctgcccca gtacaaacca gttaatccag aaagtctgca ttggagagaa 3301caatgtgata gaagaagaaa tccgtgtgaa cagaagcgtg catgagtggg caggaggcgg 3361aggaggaggg ggtggagcca cctacgtatt taagatgaag gatggagtgc cggtgcccct 3421gatcattgca gccggaggtg gtggcagggc ctacggggcc aagacagaca cgttccaccc 3481agagagactg gagaataact cctcggttct agggctaaac ggcaattccg gagccgcagg 3541tggtggaggt ggctggaatg ataacacttc cttgctctgg gccggaaaat ctttgcagga 3601gggtgccacc ggaggacatt cctgccccca ggccatgaag aagtgggggt gggagacaag 3661agggggtttc ggagggggtg gaggggggtg ctcctcaggt ggaggaggcg gaggatatat 3721aggcggcaat gcagcctcaa acaatgaccc cgaaatggat ggggaagatg gggtttcctt 3781catcagtcca ctgggcatcc tgtacacccc agctttaaaa gtgatggaag gccacgggga 3841agtgaatatt aagcattatc taaactgcag tcactgtgag gtagacgaat gtcacatgga 3901ccctgaaagc cacaaggtca tctgcttctg tgaccacggg acggtgctgg ctgaggatgg 3961cgtctcctgc attgtgtcac ccaccccgga gccacacctg ccactctcgc tgatcctctc 4021tgtggtgacc tctgccctcg tggccgccct ggtcctggct ttctccggca tcatgattgt 4081gtaccgccgg aagcaccagg agctgcaagc catgcagatg gagctgcaga gccctgagta 4141caagctgagc aagctccgca cctcgaccat catgaccgac tacaacccca actactgctt 4201tgctggcaag acctcctcca tcagtgacct gaaggaggtg ccgcggaaaa acatcaccct 4261cattcggggt ctgggccatg gcgcctttgg ggaggtgtat gaaggccagg tgtccggaat 4321gcccaacgac ccaagccccc tgcaagtggc tgtgaagacg ctgcctgaag tgtgctctga 4381acaggacgaa ctggatttcc tcatggaagc cctgatcatc agcaaattca accaccagaa 4441cattgttcgc tgcattgggg tgagcctgca atccctgccc cggttcatcc tgctggagct 4501catggcgggg ggagacctca agtccttcct ccgagagacc cgccctcgcc cgagccagcc 4561ctcctccctg gccatgctgg accttctgca cgtggctcgg gacattgcct gtggctgtca 4621gtatttggag gaaaaccact tcatccaccg agacattgct gccagaaact gcctcttgac 4681ctgtccaggc cctggaagag tggccaagat tggagacttc gggatggccc gagacatcta 4741cagggcgagc tactatagaa agggaggctg tgccatgctg ccagttaagt ggatgccccc 4801agaggccttc atggaaggaa tattcacttc taaaacagac acatggtcct ttggagtgct 4861gctatgggaa atcttttctc ttggatatat gccatacccc agcaaaagca accaggaagt 4921tctggagttt gtcaccagtg gaggccggat ggacccaccc aagaactgcc ctgggcctgt 4981ataccggata atgactcagt gctggcaaca tcagcctgaa gacaggccca actttgccat 5041cattttggag aggattgaat actgcaccca ggacccggat gtaatcaaca ccgctttgcc 5101gatagaatat ggtccacttg tggaagagga agagaaagtg cctgtgaggc ccaaggaccc 5161tgagggggtt cctcctctcc tggtctctca acaggcaaaa cgggaggagg agcgcagccc 5221agctgcccca ccacctctgc ctaccacctc ctctggcaag gctgcaaaga aacccacagc 5281tgcagagatc tctgttcgag tccctagagg gccggccgtg gaagggggac acgtgaatat 5341ggcattctct cagtccaacc ctccttcgga gttgcacaag gtccacggat ccagaaacaa
5401gcccaccagc ttgtggaacc caacgtacgg ctcctggttt acagagaaac ccaccaaaaa
5461gaataatcct atagcaaaga aggagccaca cgacaggggt aacctggggc tggagggaag
5521ctgtactgtc ccacctaacg ttgcaactgg gagacttccg ggggcctcac tgctcctaga
5581gccctcttcg ctgactgcca atatgaagga ggtacctctg ttcaggctac gtcacttccc
5641ttgtgggaat gtcaattacg gctaccagca acagggcttg cccttagaag ccgctactgc
5701ccctggagct ggtcattacg aggataccat tctgaaaagc aagaatagca tgaaccagcc
5761tgggccctga gctcggtcgc acactcactt ctcttccttg ggatccctaa gaccgtggag
5821gagagagagg caatggctcc ttcacaaacc agagaccaaa tgtcacgttt tgttttgtgc
5881caacctattt tgaagtacca ccaaaaaagc tgtattttga aaatgcttta gaaaggtttt
5941gagcatgggt tcatcctatt ctttcgaaag aagaaaatat cataaaaatg agtgataaat
6001acaaggccca gatgtggttg cataaggttt ttatgcatgt ttgttgtata cttccttatg
6061cttctttcaa attgtgtgtg ctctgcttca atgtagtcag aattagctgc ttctatgttt
6121catagttggg gtcatagatg tttccttgcc ttgttgatgt ggacatgagc catttgaggg
6181gagagggaac ggaaataaag gagttatttg taatgactaa aa
Wild type cDNA sequence TGC (4373 to the 4375) coding amino acid whose codon mutation in addition to cysteine or With a kind of same corresponding sudden change.
Wild type cDNA sequence CTG (4493 to the 4495) coding amino acid whose codon mutation in addition to leucine or with A kind of same corresponding sudden change.
The sudden change of wild type cDNA sequence G4374A or a kind of same corresponding sudden change.
The sudden change of wild type cDNA sequence G4493A or a kind of same corresponding sudden change.
Wild type alk protein matter sequence (NP_004295.2；GI:29029632):
1mgaigllwll plllstaavg sgmgtgqrag spaagpplqp replsysrlq rkslavdfvv
61pslfrvyard lllppsssel kagrpeargs laldcapllr llgpapgvsw tagspapaea
121rtlsrvlkgg svrklrrakq lvlelgeeai legcvgppge aavgllqfnl selfswwirq
181gegrlrirlm pekkasevgr egrlsaaira sqprllfqif gtghsslesp tnmpspspdy
241ftwnltwimk dsfpflshrs ryglecsfdf pceleysppl hdlrnqswsw rripseeasq
301mdlldgpgae rskemprgsf lllntsadsk htilspwmrs ssehctlavs vhrhlqpsgr
361yiaqllphne aareillmpt pgkhgwtvlq grigrpdnpf rvaleyissg nrslsavdff
421alkncsegts pgskmalqss ftcwngtvlq lgqacdfhqd caqgedesqm crklpvgfyc
481nfedgfcgwt qgtlsphtpq wqvrtlkdar fqdhqdhall lsttdvpase satvtsatfp
541apiksspcel rmswlirgvl rgnvslvlve nktgkeqgrm vwhvaayegl slwqwmvlpl
601ldvsdrfwlq mvawwgqgsr aivafdnisi sldcyltisg edkilqntap ksrnlfernp
661nkelkpgens prqtpifdpt vhwlfttcga sgphgptqaq cnnayqnsnl svevgsegpl
721kgiqiwkvpa tdtysisgyg aaggkggknt mmrshgvsvl gifnlekddm lyilvgqqge
781dacpstnqli qkvcigennv ieeeirvnrs vhewaggggg gggatyvfkm kdgvpvplii
841aaggggrayg aktdtfhper lennssvlgl ngnsgaaggg ggwndntsll wagkslqega
901tgghscpqam kkwgwetrgg fggggggcss ggggggyigg naasnndpem dgedgvsfis
961plgilytpal kvmeghgevn ikhylncshc evdechmdpe shkvicfcdh gtvlaedgvs
1021civsptpeph lplslilsvv tsalvaalvl afsgimivyr rkhqelqamq melqspeykl
1081sklrtstimt dynpnycfag ktssisdlke vprknitlir glghgafgev yegqvsgmpn
1141dpsplqvavk tlpevcseqd eldflmeali iskfnhqniv rcigvslqsl prfillelma
1201ggdlksflre trprpsqpss lamldllhva rdiacgcqyl eenhfihrdi aarnclltcp
1261gpgrvakigd fgmardiyra syyrkggcam lpvkwmppea fmegiftskt dtwsfgvllw
1321eifslgympy psksnqevle fvtsggrmdp pkncpgpvyr imtqcwqhqp edrpnfaiil
1381erieyctqdp dvintalpie ygplveeeek vpvrpkdpeg vppllvsqqa kreeerspaa
1441ppplpttssg kaakkptaae isvrvprgpa vegghvnmaf sqsnppselh kvhgsrnkpt
1501slwnptygsw ftekptkknn piakkephdr gnlglegsct vppnvatgrl pgasllleps
1561sltanmkevp lfrlrhfpcg nvnygyqqqg lpleaatapg aghyedtilk sknsmnqpgp
Wild-type protein sequence Cys1156Xaa suddenlys change, wherein Xaa aminoacid except a kind of cysteine in addition to or a kind of and Identical corresponding sudden change.
Wild-type protein sequence Leu1196Xaa suddenlys change, and wherein Xaa aminoacid in addition to a kind of leucine or one are therewith Identical corresponding sudden change.
Wild-type protein sequence Cys1156Tyr sudden change or a kind of same corresponding sudden change.
Wild-type protein sequence Leu1196Met sudden change or a kind of same corresponding sudden change.
EML4-ALK sudden change 1cDNA sequence (AB274722.1；GI:152002652)
1ggcggcgcgg cgcggcgctc gcggctgctg cctgggaggg aggccgggca ggcggctgag
61cggcgcggct ctcaacgtga cggggaagtg gttcgggcgg ccgcggctta ctaccccagg
121gcgaacggac ggacgacgga ggcgggagcc ggtagccgag ccgggcgacc tagagaacga 181gcgggtcagg ctcagcgtcg gccactctgt cggtccgctg aatgaagtgc ccgcccctct 241gagcccggag cccggcgctt tccccgcaag atggacggtt tcgccggcag tctcgatgat 301agtatttctg ctgcaagtac ttctgatgtt caagatcgcc tgtcagctct tgagtcacga 361gttcagcaac aagaagatga aatcactgtg ctaaaggcgg ctttggctga tgttttgagg 421cgtcttgcaa tctctgaaga tcatgtggcc tcagtgaaaa aatcagtctc aagtaaaggc 481caaccaagcc ctcgagcagt tattcccatg tcctgtataa ccaatggaag tggtgcaaac 541agaaaaccaa gtcataccag tgctgtctca attgcaggaa aagaaactct ttcatctgct 601gctaaaagtg gtacagaaaa aaagaaagaa aaaccacaag gacagagaga aaaaaaagag 661gaatctcatt ctaatgatca aagtccacaa attcgagcat caccttctcc ccagccctct 721tcacaacctc tccaaataca cagacaaact ccagaaagca agaatgctac tcccaccaaa 781agcataaaac gaccatcacc agctgaaaag tcacataatt cttgggaaaa ttcagatgat 841agccgtaata aattgtcgaa aataccttca acacccaaat taataccaaa agttaccaaa 901actgcagaca agcataaaga tgtcatcatc aaccaagaag gagaatatat taaaatgttt 961atgcgcggtc ggccaattac catgttcatt ccttccgatg ttgacaacta tgatgacatc 1021agaacggaac tgcctcctga gaagctcaaa ctggagtggg catatggtta tcgaggaaag 1081gactgtagag ctaatgttta ccttcttccg accggggaaa tagtttattt cattgcatca 1141gtagtagtac tatttaatta tgaggagaga actcagcgac actacctggg ccatacagac 1201tgtgtgaaat gccttgctat acatcctgac aaaattagga ttgcaactgg acagatagct 1261ggcgtggata aagatggaag gcctctacaa ccccacgtca gagtgtggga ttctgttact 1321ctatccacac tgcagattat tggacttggc acttttgagc gtggagtagg atgcctggat 1381ttttcaaaag cagattcagg tgttcattta tgtgttattg atgactccaa tgagcatatg 1441cttactgtat gggactggca gaagaaagca aaaggagcag aaataaagac aacaaatgaa 1501gttgttttgg ctgtggagtt tcacccaaca gatgcaaata ccataattac atgcggtaaa 1561tctcatattt tcttctggac ctggagcggc aattcactaa caagaaaaca gggaattttt 1621gggaaatatg aaaagccaaa atttgtgcag tgtttagcat tcttggggaa tggagatgtt 1681cttactggag actcaggtgg agtcatgctt atatggagca aaactactgt agagcccaca 1741cctgggaaag gacctaaagt gtaccgccgg aagcaccagg agctgcaagc catgcagatg 1801gagctgcaga gccctgagta caagctgagc aagctccgca cctcgaccat catgaccgac 1861tacaacccca actactgctt tgctggcaag acctcctcca tcagtgacct gaaggaggtg 1921ccgcggaaaa acatcaccct cattcggggt ctgggccatg gagcctttgg ggaggtgtat 1981gaaggccagg tgtccggaat gcccaacgac ccaagccccc tgcaagtggc tgtgaagacg 2041ctgcctgaag tgtgctctga acaggacgaa ctggatttcc tcatggaagc cctgatcatc 2101agcaaattca accaccagaa cattgttcgc tgcattgggg tgagcctgca atccctgccc 2161cggttcatcc tgctggagct catggcgggg ggagacctca agtccttcct ccgagagacc 2221cgccctcgcc cgagccagcc ctcctccctg gccatgctgg accttctgca cgtggctcgg 2281gacattgcct gtggctgtca gtatttggag gaaaaccact tcatccaccg agacattgct 2341gccagaaact gcctcttgac ctgtccaggc cctggaagag tggccaagat tggagacttc 2401gggatggccc gagacatcta cagggcgagc tactatagaa agggaggctg tgccatgctg 2461ccagttaagt ggatgccccc agaggccttc atggaaggaa tattcacttc taaaacagac 2521acatggtcct ttggagtgct gctatgggaa atcttttctc ttggatatat gccatacccc 2581agcaaaagca accaggaagt tctggagttt gtcaccagtg gaggccggat ggacccaccc 2641aagaactgcc ctgggcctgt ataccggata atgactcagt gctggcaaca tcagcctgaa 2701gacaggccca actttgccat cattttggag aggattgaat actgcaccca ggacccggat 2761gtaatcaaca ccgctttgcc gatagaatat ggtccacttg tggaagagga agagaaagtg 2821cctgtgaggc ccaaggaccc tgagggggtt cctcctctcc tggtctctca acaggcaaaa 2881cgggaggagg agcgcagccc agctgcccca ccacctctgc ctaccacctc ctctggcaag 2941gctgcaaaga aacccacagc tgcagaggtc tctgttcgag tccctagagg gccggccgtg 3001gaagggggac acgtgaatat ggcattctct cagtccaacc ctccttcgga gttgcacagg 3061gtccacggat ccagaaacaa gcccaccagc ttgtggaacc caacgtacgg ctcctggttt 3121acagagaaac ccaccaaaaa gaataatcct atagcaaaga aggagccaca cgagaggggt 3181aacctggggc tggagggaag ctgtactgtc ccacctaacg ttgcaactgg gagacttccg 3241ggggcctcac tgctcctaga gccctcttcg ctgactgcca atatgaagga ggtacctctg 3301ttcaggctac gtcacttccc ttgtgggaat gtcaattacg gctaccagca acagggcttg 3361cccttagaag ccgctactgc ccctggagct ggtcattacg aggataccat tctgaaaagc 3421aagaatagca tgaaccagcc tgggccctga gctcggtcac acactcactt ctcttccttg 3481ggatccctaa gaccgtggag gagagagagg caatcaatgg ctccttcaca aaccagagac 3541caaatgtcac gttttgtttt gtgccaacct attttgaagt accaccaaaa aagctgtatt 3601ttgaaaatgc tttagaaagg ttttgagcat gggttcatcc tattctttcg aaagaagaaa 3661atatcataaa aatgagtgat aaatacaagg cccagatgtg gttgcataag gtttttatgc 3721atgtttgttg tatacttcct tatgcttctt ttaaattgtg tgtgctctgc ttcaatgtag 3781tcagaattag ctgcttctat gtttcatagt tggggtcata gatgtttcct tgccttgttg 3841atgtggacat gagccatttg aggggagagg gaacggaaat aaaggagtta tttgtaatga 3901aaaaaaaaaa aaaaaaaaaa aaaaaa
EML4-ALK suddenly change 1 protein sequence (BAF73611.1；GI:152002653)
1mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva
61svkksvsskg qpspravipm scitngsgan rkpshtsavs iagketlssa aksgtekkke
121kpqgqrekke eshsndqspq iraspspqps sqplqihrqt pesknatptk sikrpspaek 181shnswensdd srnklskips tpklipkvtk tadkhkdvii nqegeyikmf mrgrpitmfi 241psdvdnyddi rtelppeklk lewaygyrgk dcranvyllp tgeivyfias vvvlfnyeer 301tqrhylghtd cvkclaihpd kiriatgqia gvdkdgrplq phvrvwdsvt lstlqiiglg 361tfergvgcld fskadsgvhl cviddsnehm ltvwdwqkka kgaeikttne vvlavefhpt 421dantiitcgk shiffwtwsg nsltrkqgif gkyekpkfvq claflgngdv ltgdsggvml 481iwskttvept pgkgpkvyrr khqelqamqm elqspeykls klrtstimtd ynpnycfagk 541tssisdlkev prknitlirg lghgafgevy egqvsgmpnd psplqvavkt lpevcseqde 601ldflmealii skfnhqnivr cigvslqslp rfillelmag gdlksflret rprpsqpssl 661amldllhvar diacgcqyle enhfihrdia arnclltcpg pgrvakigdf gmardiyras 721yyrkggcaml pvkwmppeaf megiftsktd twsfgvllwe ifslgympyp sksnqevlef 781vtsggrmdpp kncpgpvyri mtqcwqhqpe drpnfaiile rieyctqdpd vintalpiey 841gplveeeekv pvrpkdpegv ppllvsqqak reeerspaap pplpttssgk aakkptaaev 901svrvprgpav egghvnmafs qsnppselhr vhgsrnkpts lwnptygswf tekptkknnp 961iakkepherg nlglegsctv ppnvatgrlp gaslllepss ltanmkevpl frlrhfpcgn 1021vnygyqqqgl pleaatapga ghyedtilks knsmnqpgp
EML4-ALK sudden change 2cDNA sequence (AB275889.1；GI:152002654)
1ggcggcgcgg cgcggcgctc gcggctgctg cctgggaggg aggccgggca ggcggctgag
61cggcgcggct ctcaacgtga cggggaagtg gttcgggcgg ccgcggctta ctaccccagg
121gcgaacggac ggacgacgga ggcgggagcc ggtagccgag ccgggcgacc tagagaacga 181gcgggtcagg ctcagcgtcg gccactctgt cggtccgctg aatgaagtgc ccgcccctct 241gagcccggag cccggcgctt tccccgcaag atggacggtt tcgccggcag tctcgatgat 301agtatttctg ctgcaagtac ttctgatgtt caagatcgcc tgtcagctct tgagtcacga 361gttcagcaac aagaagatga aatcactgtg ctaaaggcgg ctttggctga tgttttgagg 421cgtcttgcaa tctctgaaga tcatgtggcc tcagtgaaaa aatcagtctc aagtaaaggc 481caaccaagcc ctcgagcagt tattcccatg tcctgtataa ccaatggaag tggtgcaaac 541agaaaaccaa gtcataccag tgctgtctca attgcaggaa aagaaactct ttcatctgct 601gctaaaagtg gtacagaaaa aaagaaagaa aaaccacaag gacagagaga aaaaaaagag 661gaatctcatt ctaatgatca aagtccacaa attcgagcat caccttctcc ccagccctct 721tcacaacctc tccaaataca cagacaaact ccagaaagca agaatgctac tcccaccaaa 781agcataaaac gaccatcacc agctgaaaag tcacataatt cttgggaaaa ttcagatgat 841agccgtaata aattgtcgaa aataccttca acacccaaat taataccaaa agttaccaaa 901actgcagaca agcataaaga tgtcatcatc aaccaagaag gagaatatat taaaatgttt 961atgcgcggtc ggccaattac catgttcatt ccttccgatg ttgacaacta tgatgacatc 1021agaacggaac tgcctcctga gaagctcaaa ctggagtggg catatggtta tcgaggaaag 1081gactgtagag ctaatgttta ccttcttccg accggggaaa tagtttattt cattgcatca 1141gtagtagtac tatttaatta tgaggagaga actcagcgac actacctggg ccatacagac 1201tgtgtgaaat gccttgctat acatcctgac aaaattagga ttgcaactgg acagatagct 1261ggcgtggata aagatggaag gcctctacaa ccccacgtca gagtgtggga ttctgttact 1321ctatccacac tgcagattat tggacttggc acttttgagc gtggagtagg atgcctggat 1381ttttcaaaag cagattcagg tgttcattta tgtgttattg atgactccaa tgagcatatg 1441cttactgtat gggactggca gaagaaagca aaaggagcag aaataaagac aacaaatgaa 1501gttgttttgg ctgtggagtt tcacccaaca gatgcaaata ccataattac atgcggtaaa 1561tctcatattt tcttctggac ctggagcggc aattcactaa caagaaaaca gggaattttt 1621gggaaatatg aaaagccaaa atttgtgcag tgtttagcat tcttggggaa tggagatgtt 1681cttactggag actcaggtgg agtcatgctt atatggagca aaactactgt agagcccaca 1741cctgggaaag gacctaaagg tgtatatcaa atcagcaaac aaatcaaagc tcatgatggc 1801agtgtgttca cactttgtca gatgagaaat gggatgttat taactggagg agggaaagac 1861agaaaaataa ttctgtggga tcatgatctg aatcctgaaa gagaaataga ggttcctgat 1921cagtatggca caatcagagc tgtagcagaa ggaaaggcag atcaattttt agtaggcaca 1981tcacgaaact ttattttacg aggaacattt aatgatggct tccaaataga agtacagggt 2041catacagatg agctttgggg tcttgccaca catcccttca aagatttgct cttgacatgt 2101gctcaggaca ggcaggtgtg cctgtggaac tcaatggaac acaggctgga atggaccagg 2161ctggtagatg aaccaggaca ctgtgcagat tttcatccaa gtggcacagt ggtggccata 2221ggaacgcact caggcaggtg gtttgttctg gatgcagaaa ccagagatct agtttctatc 2281cacacagacg ggaatgaaca gctctctgtg atgcgctact caatagatgg taccttcctg 2341gctgtaggat ctcatgacaa ctttatttac ctctatgtag tctctgaaaa tggaagaaaa 2401tatagcagat atggaaggtg cactggacat tccagctaca tcacacacct tgactggtcc 2461ccagacaaca agtatataat gtctaactcg ggagactatg aaatattgta cttgtaccgc 2521cggaagcacc aggagctgca agccatgcag atggagctgc agagccctga gtacaagctg 2581agcaagctcc gcacctcgac catcatgacc gactacaacc ccaactactg ctttgctggc 2641aagacctcct ccatcagtga cctgaaggag gtgccgcgga aaaacatcac cctcattcgg 2701ggtctgggcc atggagcctt tggggaggtg tatgaaggcc aggtgtccgg aatgcccaac 2761gacccaagcc ccctgcaagt ggctgtgaag acgctgcctg aagtgtgctc tgaacaggac 2821gaactggatt tcctcatgga agccctgatc atcagcaaat tcaaccacca gaacattgtt 2881cgctgcattg gggtgagcct gcaatccctg ccccggttca tcctgctgga gctcatggcg 2941gggggagacc tcaagtcctt cctccgagag acccgccctc gcccgagcca gccctcctcc 3001ctggccatgc tggaccttct gcacgtggct cgggacattg cctgtggctg tcagtatttg 3061gaggaaaacc acttcatcca ccgagacatt gctgccagaa actgcctctt gacctgtcca 3121ggccctggaa gagtggccaa gattggagac ttcgggatgg cccgagacat ctacagggcg 3181agctactata gaaagggagg ctgtgccatg ctgccagtta agtggatgcc cccagaggcc 3241ttcatggaag gaatattcac ttctaaaaca gacacatggt cctttggagt gctgctatgg 3301gaaatctttt ctcttggata tatgccatac cccagcaaaa gcaaccagga agttctggag 3361tttgtcacca gtggaggccg gatggaccca cccaagaact gccctgggcc tgtataccgg 3421ataatgactc agtgctggca acatcagcct gaagacaggc ccaactttgc catcattttg 3481gagaggattg aatactgcac ccaggacccg gatgtaatca acaccgcttt gccgatagaa 3541tatggtccac ttgtggaaga ggaagagaaa gtgcctgtga ggcccaagga ccctgagggg 3601gttcctcctc tcctggtctc tcaacaggca aaacgggagg aggagcgcag cccagctgcc 3661ccaccacctc tgcctaccac ctcctctggc aaggctgcaa agaaacccac agctgcagag 3721gtctctgttc gagtccctag agggccggcc gtggaagggg gacacgtgaa tatggcattc 3781tctcagtcca accctccttc ggagttgcac agggtccacg gatccagaaa caagcccacc 3841agcttgtgga acccaacgta cggctcctgg tttacagaga aacccaccaa aaagaataat 3901cctatagcaa agaaggagcc acacgagagg ggtaacctgg ggctggaggg aagctgtact 3961gtcccaccta acgttgcaac tgggagactt ccgggggcct cactgctcct agagccctct 4021tcgctgactg ccaatatgaa ggaggtacct ctgttcaggc tacgtcactt cccttgtggg 4081aatgtcaatt acggctacca gcaacagggc ttgcccttag aagccgctac tgcccctgga 4141gctggtcatt acgaggatac cattctgaaa agcaagaata gcatgaacca gcctgggccc 4201tgagctcggt cacacactca cttctcttcc ttgggatccc taagaccgtg gaggagagag 4261aggcaatcaa tggctccttc acaaaccaga gaccaaatgt cacgttttgt tttgtgccaa 4321cctattttga agtaccacca aaaaagctgt attttgaaaa tgctttagaa aggttttgag 4381catgggttca tcctattctt tcgaaagaag aaaatatcat aaaaatgagt gataaataca 4441aggcccagat gtggttgcat aaggttttta tgcatgtttg ttgtatactt ccttatgctt 4501cttttaaatt gtgtgtgctc tgcttcaatg tagtcagaat tagctgcttc tatgtttcat 4561agttggggtc atagatgttt ccttgccttg ttgatgtgga catgagccat ttgaggggag 4621agggaacgga aataaaggag ttatttgtaa tgaaaaaaaa aaaaaaaaaa aaaaaaaaa
EML4-ALK suddenly change 2 protein sequences (BAF73612.1；GI:152002655)
1mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva
61svkksvsskg qpspravipm scitngsgan rkpshtsavs iagketlssa aksgtekkke
121kpqgqrekke eshsndqspq iraspspqps sqplqihrqt pesknatptk sikrpspaek 181shnswensdd srnklskips tpklipkvtk tadkhkdvii nqegeyikmf mrgrpitmfi 241psdvdnyddi rtelppeklk lewaygyrgk dcranvyllp tgeivyfias vvvlfnyeer 301tqrhylghtd cvkclaihpd kiriatgqia gvdkdgrplq phvrvwdsvt lstlqiiglg 361tfergvgcld fskadsgvhl cviddsnehm ltvwdwqkka kgaeikttne vvlavefhpt 421dantiitcgk shiffwtwsg nsltrkqgif gkyekpkfvq claflgngdv ltgdsggvml 481iwskttvept pgkgpkgvyq iskqikahdg svftlcqmrn gmlltgggkd rkiilwdhdl 541npereievpd qygtiravae gkadqflvgt srnfilrgtf ndgfqievqg htdelwglat 601hpfkdllltc aqdrqvclwn smehrlewtr lvdepghcad fhpsgtvvai gthsgrwfvl 661daetrdlvsi htdgneqlsv mrysidgtfl avgshdnfiy lyvvsengrk ysrygrctgh 721ssyithldws pdnkyimsns gdyeilylyr rkhqelqamq melqspeykl sklrtstimt 781dynpnycfag ktssisdlke vprknitlir glghgafgev yegqvsgmpn dpsplqvavk 841tlpevcseqd eldflmeali iskfnhqniv rcigvslqsl prfillelma ggdlksflre 901trprpsqpss lamldllhva rdiacgcqyl eenhfihrdi aarnclltcp gpgrvakigd 961fgmardiyra syyrkggcam lpvkwmppea fmegiftskt dtwsfgvllw eifslgympy 1021psksnqevle fvtsggrmdp pkncpgpvyr imtqcwqhqp edrpnfaiil erieyctqdp 1081dvintalpie ygplveeeek vpvrpkdpeg vppllvsqqa kreeerspaa ppplpttssg 1141kaakkptaae vsvrvprgpa vegghvnmaf sqsnppselh rvhgsrnkpt slwnptygsw 1201ftekptkknn piakkepher gnlglegsct vppnvatgrl pgasllleps sltanmkevp 1261lfrlrhfpcg nvnygyqqqg lpleaatapg aghyedtilk sknsmnqpgp
EML4-ALK sudden change 3a nucleotide sequence (AB374361.1；GI:194072592)
1actctgtcgg tccgctgaat gaagtgcccg cccctctaag cccggagccc ggcgctttcc
61ccgcaagatg gacggtttcg ccggcagtct cgatgatagt atttctgctg caagtacttc
121tgatgttcaa gatcgcctgt cagctcttga gtcacgagtt cagcaacaag aagatgaaat 181cactgtgcta aaggcggctt tggctgatgt tttgaggcgt cttgcaatct ctgaagatca 241tgtggcctca gtgaaaaaat cagtctcaag taaaggccaa ccaagccctc gagcagttat 301tcccatgtcc tgtataacca atggaagtgg tgcaaacaga aaaccaagtc ataccagtgc 361tgtctcaatt gcaggaaaag aaactctttc atctgctgct aaaagtggta cagaaaaaaa 421gaaagaaaaa ccacaaggac agagagaaaa aaaagaggaa tctcattcta atgatcaaag 481tccacaaatt cgagcatcac cttctcccca gccctcttca caacctctcc aaatacacag 541acaaactcca gaaagcaaga atgctactcc caccaaaagc ataaaacgac catcaccagc 601tgaaaagtca cataattctt gggaaaattc agatgatagc cgtaataaat tgtcgaaaat 661accttcaaca cccaaattaa taccaaaagt taccaaaact gcagacaagc ataaagatgt 721catcatcaac caagtgtacc gccggaagca ccaggagctg caagccatgc agatggagct 781gcagagccct gagtacaagc tgagcaagct ccgcacctcg accatcatga ccgactacaa 841ccccaactac tgctttgctg gcaagacctc ctccatcagt gacctgaagg aggtgccgcg 901gaaaaacatc accctcattc ggggtctggg ccatggagcc tttggggagg tgtatgaagg 961ccaggtgtcc ggaatgccca acgacccaag ccccctgcaa gtggctgtga agacgctgcc 1021tgaagtgtgc tctgaacagg acgaactgga tttcctcatg gaagccctga tcatcagcaa 1081attcaaccac cagaacattg ttcgctgcat tggggtgagc ctgcaatccc tgccccggtt 1141catcctgctg gagctcatgg cggggggaga cctcaagtcc ttcctccgag agacccgccc 1201tcgcccgagc cagccctcct ccctggccat gctggacctt ctgcacgtgg ctcgggacat 1261tgcctgtggc tgtcagtatt tggaggaaaa ccacttcatc caccgagaca ttgctgccag 1321aaactgcctc ttgacctgtc caggccctgg aagagtggcc aagattggag acttcgggat 1381ggcccgagac atctacaggg cgagctacta tagaaaggga ggctgtgcca tgctgccagt 1441taagtggatg cccccagagg ccttcatgga aggaatattc acttctaaaa cagacacatg 1501gtcctttgga gtgctgctat gggaaatctt ttctcttgga tatatgccat accccagcaa 1561aagcaaccag gaagttctgg agtttgtcac cagtggaggc cggatggacc cacccaagaa 1621ctgccctggg cctgtatacc ggataatgac tcagtgctgg caacatcagc ctgaagacag 1681gcccaacttt gccatcattt tggagaggat tgaatactgc acccaggacc cggatgtaat 1741caacaccgct ttgccgatag aatatggtcc acttgtggaa gaggaagaga aagtgcctgt 1801gaggcccaag gaccctgagg gggttcctcc tctcctggtc tctcaacagg caaaacggga 1861ggaggagcgc agcccagctg ccccaccacc tctgcctacc acctcctctg gcaaggctgc 1921aaagaaaccc acagctgcag aggtctctgt tcgagtccct agagggccgg ccgtggaagg 1981gggacacgtg aatatggcat tctctcagtc caaccctcct tcggagttgc acagggtcca 2041cggatccaga aacaagccca ccagcttgtg gaacccaacg tacggctcct ggtttacaga 2101gaaacccacc aaaaagaata atcctatagc aaagaaggag ccacacgaga ggggtaacct 2161ggggctggag ggaagctgta ctgtcccacc taacgttgca actgggagac ttccgggggc 2221ctcactgctc ctagagccct cttcgctgac tgccaatatg aaggaggtac ctctgttcag 2281gctacgtcac ttcccttgtg ggaatgtcaa ttacggctac cagcaacagg gcttgccctt 2341agaagccgct actgcccctg gagctggtca ttacgaggat accattctga aaagcaagaa 2401tagcatgaac cagcctgggc cctgagctcg gtcgcacact cacttctctt ccttgggatc 2461cctaagaccg tgg
EML4-ALK sudden change 3a protein sequence (BAG55003.1；GI:194072593)
1mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva
61svkksvsskg qpspravipm scitngsgan rkpshtsavs iagketlssa aksgtekkke
121kpqgqrekke eshsndqspq iraspspqps sqplqihrqt pesknatptk sikrpspaek 181shnswensdd srnklskips tpklipkvtk tadkhkdvii nqvyrrkhqe lqamqmelqs 241peyklsklrt stimtdynpn ycfagktssi sdlkevprkn itlirglghg afgevyegqv 301sgmpndpspl qvavktlpev cseqdeldfl mealiiskfn hqnivrcigv slqslprfil 361lelmaggdlk sflretrprp sqpsslamld llhvardiac gcqyleenhf ihrdiaarnc 421lltcpgpgrv akigdfgmar diyrasyyrk ggcamlpvkw mppeafmegi ftsktdtwsf 481gvllweifsl gympypsksn qevlefvtsg grmdppkncp gpvyrimtqc wqhqpedrpn 541faiileriey ctqdpdvint alpieygplv eeeekvpvrp kdpegvppll vsqqakreee 601rspaappplp ttssgkaakk ptaaevsvrv prgpaveggh vnmafsqsnp pselhrvhgs 661rnkptslwnp tygswftekp tkknnpiakk ephergnlgl egsctvppnv atgrlpgasl 721llepssltan mkevplfrlr hfpcgnvnyg yqqqglplea atapgaghye dtilksknsm 781nqpgp
EML4-ALK sudden change 3b nucleotide sequence (AB374362.1；GI:194072594)
1actctgtcgg tccgctgaat gaagtgcccg cccctctaag cccggagccc ggcgctttcc
61ccgcaagatg gacggtttcg ccggcagtct cgatgatagt atttctgctg caagtacttc
121tgatgttcaa gatcgcctgt cagctcttga gtcacgagtt cagcaacaag aagatgaaat 181cactgtgcta aaggcggctt tggctgatgt tttgaggcgt cttgcaatct ctgaagatca 241tgtggcctca gtgaaaaaat cagtctcaag taaaggccaa ccaagccctc gagcagttat 301tcccatgtcc tgtataacca atggaagtgg tgcaaacaga aaaccaagtc ataccagtgc 361tgtctcaatt gcaggaaaag aaactctttc atctgctgct aaaagtggta cagaaaaaaa 421gaaagaaaaa ccacaaggac agagagaaaa aaaagaggaa tctcattcta atgatcaaag 481tccacaaatt cgagcatcac cttctcccca gccctcttca caacctctcc aaatacacag 541acaaactcca gaaagcaaga atgctactcc caccaaaagc ataaaacgac catcaccagc 601tgaaaagtca cataattctt gggaaaattc agatgatagc cgtaataaat tgtcgaaaat 661accttcaaca cccaaattaa taccaaaagt taccaaaact gcagacaagc ataaagatgt 721catcatcaac caagcaaaaa tgtcaactcg cgaaaaaaac agccaagtgt accgccggaa 781gcaccaggag ctgcaagcca tgcagatgga gctgcagagc cctgagtaca agctgagcaa 841gctccgcacc tcgaccatca tgaccgacta caaccccaac tactgctttg ctggcaagac 901ctcctccatc agtgacctga aggaggtgcc gcggaaaaac atcaccctca ttcggggtct 961gggccatgga gcctttgggg aggtgtatga aggccaggtg tccggaatgc ccaacgaccc 1021aagccccctg caagtggctg tgaagacgct gcctgaagtg tgctctgaac aggacgaact 1081ggatttcctc atggaagccc tgatcatcag caaattcaac caccagaaca ttgttcgctg 1141cattggggtg agcctgcaat ccctgccccg gttcatcctg ctggagctca tggcgggggg 1201agacctcaag tccttcctcc gagagacccg ccctcgcccg agccagccct cctccctggc 1261catgctggac cttctgcacg tggctcggga cattgcctgt ggctgtcagt atttggagga 1321aaaccacttc atccaccgag acattgctgc cagaaactgc ctcttgacct gtccaggccc 1381tggaagagtg gccaagattg gagacttcgg gatggcccga gacatctaca gggcgagcta 1441ctatagaaag ggaggctgtg ccatgctgcc agttaagtgg atgcccccag aggccttcat 1501ggaaggaata ttcacttcta aaacagacac atggtccttt ggagtgctgc tatgggaaat 1561cttttctctt ggatatatgc cataccccag caaaagcaac caggaagttc tggagtttgt 1621caccagtgga ggccggatgg acccacccaa gaactgccct gggcctgtat accggataat 1681gactcagtgc tggcaacatc agcctgaaga caggcccaac tttgccatca ttttggagag 1741gattgaatac tgcacccagg acccggatgt aatcaacacc gctttgccga tagaatatgg 1801tccacttgtg gaagaggaag agaaagtgcc tgtgaggccc aaggaccctg agggggttcc 1861tcctctcctg gtctctcaac aggcaaaacg ggaggaggag cgcagcccag ctgccccacc 1921acctctgcct accacctcct ctggcaaggc tgcaaagaaa cccacagctg cagaggtctc 1981tgttcgagtc cctagagggc cggccgtgga agggggacac gtgaatatgg cattctctca 2041gtccaaccct ccttcggagt tgcacagggt ccacggatcc agaaacaagc ccaccagctt 2101gtggaaccca acgtacggct cctggtttac agagaaaccc accaaaaaga ataatcctat 2161agcaaagaag gagccacacg agaggggtaa cctggggctg gagggaagct gtactgtccc 2221acctaacgtt gcaactggga gacttccggg ggcctcactg ctcctagagc cctcttcgct 2281gactgccaat atgaaggagg tacctctgtt caggctacgt cacttccctt gtgggaatgt 2341caattacggc taccagcaac agggcttgcc cttagaagcc gctactgccc ctggagctgg 2401tcattacgag gataccattc tgaaaagcaa gaatagcatg aaccagcctg ggccctgagc 2461tcggtcgcac actcacttct cttccttggg atccctaaga ccgtgg
EML4-ALK sudden change 3b protein sequence (BAG55004.1；GI:194072595)
1mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva
61svkksvsskg qpspravipm scitngsgan rkpshtsavs iagketlssa aksgtekkke
121kpqgqrekke eshsndqspq iraspspqps sqplqihrqt pesknatptk sikrpspaek 181shnswensdd srnklskips tpklipkvtk tadkhkdvii nqakmstrek nsqvyrrkhq 241elqamqmelq speyklsklr tstimtdynp nycfagktss isdlkevprk nitlirglgh 301gafgevyegq vsgmpndpsp lqvavktlpe vcseqdeldf lmealiiskf nhqnivrcig 361vslqslprfi llelmaggdl ksflretrpr psqpsslaml dllhvardia cgcqyleenh 421fihrdiaarn clltcpgpgr vakigdfgma rdiyrasyyr kggcamlpvk wmppeafmeg 481iftsktdtws fgvllweifs lgympypsks nqevlefvts ggrmdppknc pgpvyrimtq 541cwqhqpedrp nfaiilerie yctqdpdvin talpieygpl veeeekvpvr pkdpegvppl 601lvsqqakree erspaapppl pttssgkaak kptaaevsvr vprgpavegg hvnmafsqsn 661ppselhrvhg srnkptslwn ptygswftek ptkknnpiak kephergnlg legsctvppn 721vatgrlpgas lllepsslta nmkevplfrl rhfpcgnvny gyqqqglple aatapgaghy 781edtilkskns mnqpgp
EML4-ALK suddenly change 4 nucleotide sequences (AB374363.1；GI:209837703)
1actctgtcgg tccgctgaat gaagtgcccg cccctctaag cccggagccc ggcgctttcc
61ccgcaagatg gacggtttcg ccggcagtct cgatgatagt atttctgctg caagtacttc
121tgatgttcaa gatcgcctgt cagctcttga gtcacgagtt cagcaacaag aagatgaaat 181cactgtgcta aaggcggctt tggctgatgt tttgaggcgt cttgcaatct ctgaagatca 241tgtggcctca gtgaaaaaat cagtctcaag taaaggccaa ccaagccctc gagcagttat 301tcccatgtcc tgtataacca atggaagtgg tgcaaacaga aaaccaagtc ataccagtgc 361tgtctcaatt gcaggaaaag aaactctttc atctgctgct aaaagtggta cagaaaaaaa 421gaaagaaaaa ccacaaggac agagagaaaa aaaagaggaa tctcattcta atgatcaaag 481tccacaaatt cgagcatcac cttctcccca gccctcttca caacctctcc aaatacacag 541acaaactcca gaaagcaaga atgctactcc caccaaaagc ataaaacgac catcaccagc 601tgaaaagtca cataattctt gggaaaattc agatgatagc cgtaataaat tgtcgaaaat 661accttcaaca cccaaattaa taccaaaagt taccaaaact gcagacaagc ataaagatgt 721catcatcaac caagaaggag aatatattaa aatgtttatg cgcggtcggc caattaccat 781gttcattcct tccgatgttg acaactatga tgacatcaga acggaactgc ctcctgagaa 841gctcaaactg gagtgggcat atggttatcg aggaaaggac tgtagagcta atgtttacct 901tcttccgacc ggggaaatag tttatttcat tgcatcagta gtagtactat ttaattatga 961ggagagaact cagcgacact acctgggcca tacagactgt gtgaaatgcc ttgctataca 1021tcctgacaaa attaggattg caactggaca gatagctggc gtggataaag atggaaggcc 1081tctacaaccc cacgtcagag tgtgggattc tgttactcta tccacactgc agattattgg 1141acttggcact tttgagcgtg gagtaggatg cctggatttt tcaaaagcag attcaggtgt 1201tcatttatgt gttattgatg actccaatga gcatatgctt actgtatggg actggcagag 1261gaaagcaaaa ggagcagaaa taaagacaac aaatgaagtt gttttggctg tggagtttca 1321cccaacagat gcaaatacca taattacatg cggtaaatct catattttct tctggacctg 1381gagcggcaat tcactaacaa gaaaacaggg aatttttggg aaatatgaaa agccaaaatt 1441tgtgcagtgt ttagcattct tggggaatgg agatgttctt actggagact caggtggagt 1501catgcttata tggagcaaaa ctactgtaga gcccacacct gggaaaggac ctaaaggtgt 1561atatcaaatc agcaaacaaa tcaaagctca tgatggcagt gtgttcacac tttgtcagat 1621gagaaatggg atgttattaa ctggaggagg gaaagacaga aaaataattc tgtgggatca 1681tgatctgaat cctgaaagag aaatagagat atgctggatg agccctgagt acaagctgag 1741caagctccgc acctcgacca tcatgaccga ctacaacccc aactactgct ttgctggcaa 1801gacctcctcc atcagtgacc tgaaggaggt gccgcggaaa aacatcaccc tcattcgggg 1861tctgggccat ggagcctttg gggaggtgta tgaaggccag gtgtccggaa tgcccaacga 1921cccaagcccc ctgcaagtgg ctgtgaagac gctgcctgaa gtgtgctctg aacaggacga 1981actggatttc ctcatggaag ccctgatcat cagcaaattc aaccaccaga acattgttcg 2041ctgcattggg gtgagcctgc aatccctgcc ccggttcatc ctgctggagc tcatggcggg 2101gggagacctc aagtccttcc tccgagagac ccgccctcgc ccgagccagc cctcctccct 2161ggccatgctg gaccttctgc acgtggctcg ggacattgcc tgtggctgtc agtatttgga 2221ggaaaaccac ttcatccacc gagacattgc tgccagaaac tgcctcttga cctgtccagg 2281ccctggaaga gtggccaaga ttggagactt cgggatggcc cgagacatct acagggcgag 2341ctactataga aagggaggct gtgccatgct gccagttaag tggatgcccc cagaggcctt 2401catggaagga atattcactt ctaaaacaga cacatggtcc tttggagtgc tgctatggga 2461aatcttttct cttggatata tgccataccc cagcaaaagc aaccaagaag ttctggagtt 2521tgtcaccagt ggaggccgga tggacccacc caagaactgc cctgggcctg tataccggat 2581aatgactcag tgctggcaac atcagcctga agacaggccc aactttgcca tcattttgga 2641gaggattgaa tactgcaccc aggacccgga tgtaatcaac accgctttgc cgatagaata 2701tggtccactt gtggaagagg aagagaaagt gcctgtgagg cccaaggacc ctgagggggt 2761tcctcctctc ctggtctctc aacaggcaaa acgggaggag gagcgcagcc cagctgcccc 2821accacctctg cctaccacct cctctggcaa ggctgcaaag aaacccacag ctgcagaggt 2881ctctgttcga gtccctagag ggccggccgt ggaaggggga cacgtgaata tggcattctc 2941tcagtccaac cctccttcgg agttgcacag ggtccacgga tccagaaaca agcccaccag 3001cttgtggaac ccaacgtacg gctcctggtt tacagagaaa cccaccaaaa agaataatcc 3061tatagcaaag aaggagccac acgagagggg taacctgggg ctggagggaa gctgtactgt 3121cccacctaac gttgcaactg ggagacttcc gggggcctca ctgctcctag agccctcttc 3181gctgactgcc aatatgaagg aggtacctct gttcaggcta cgtcacttcc cttgtgggaa 3241tgtcaattac ggctaccagc aacagggctt gcccttagaa gccgctactg cccctggagc 3301tggtcattac gaggatacca ttctgaaaag caagaatagc atgaaccagc ctgggccctg 3361agctcggtcg cacactcact tctcttcctt gggatcccta agaccgtgg
EML4-ALK sudden change april protein sequence (BAG75147.1；GI:209837704)
1mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva
61svkksvsskg qpspravipm scitngsgan rkpshtsavs iagketlssa aksgtekkke
121kpqgqrekke eshsndqspq iraspspqps sqplqihrqt pesknatptk sikrpspaek 181shnswensdd srnklskips tpklipkvtk tadkhkdvii nqegeyikmf mrgrpitmfi 241psdvdnyddi rtelppeklk lewaygyrgk dcranvyllp tgeivyfias vvvlfnyeer 301tqrhylghtd cvkclaihpd kiriatgqia gvdkdgrplq phvrvwdsvt lstlqiiglg 361tfergvgcld fskadsgvhl cviddsnehm ltvwdwqrka kgaeikttne vvlavefhpt 421dantiitcgk shiffwtwsg nsltrkqgif gkyekpkfvq claflgngdv ltgdsggvml 481iwskttvept pgkgpkgvyq iskqikahdg svftlcqmrn gmlltgggkd rkiilwdhdl 541npereieicw mspeyklskl rtstimtdyn pnycfagkts sisdlkevpr knitlirglg 601hgafgevyeg qvsgmpndps plqvavktlp evcseqdeld flmealiisk fnhqnivrci 661gvslqslprf illelmaggd lksflretrp rpsqpsslam ldllhvardi acgcqyleen 721hfihrdiaar nclltcpgpg rvakigdfgm ardiyrasyy rkggcamlpv kwmppeafme 781giftsktdtw sfgvllweif slgympypsk snqevlefvt sggrmdppkn cpgpvyrimt 841qcwqhqpedr pnfaiileri eyctqdpdvi ntalpieygp lveeeekvpv rpkdpegvpp 901llvsqqakre eerspaappp lpttssgkaa kkptaaevsv rvprgpaveg ghvnmafsqs 961nppselhrvh gsrnkptslw nptygswfte kptkknnpia kkephergnl glegsctvpp 1021nvatgrlpga slllepsslt anmkevplfr lrhfpcgnvn ygyqqqglpl eaatapgagh 1081yedtilkskn smnqpgp
EML4-ALK sudden change 5a nucleotide sequence (AB374364.1；GI:209837705)
1actctgtcgg tccgctgaat gaagtgcccg cccctctaag cccggagccc ggcgctttcc
61ccgcaagatg gacggtttcg ccggcagtct cgatgatagt atttctgctg caagtacttc
121tgatgttcaa gatcgcctgt cagctcttga gtcacgagtt cagcaacaag aagatgaaat 181cactgtgcta aaggcggctt tggctgatgt tttgaggcgt cttgcaatct ctgaagatca 241tgtggcctca gtgaaaaaat cagtctcaag taaagtgtac cgccggaagc accaggagct 301gcaagccatg cagatggagc tgcagagccc tgagtacaag ctgagcaagc tccgcacctc 361gaccatcatg accgactaca accccaacta ctgctttgct ggcaagacct cctccatcag 421tgacctgaag gaggtgccgc ggaaaaacat caccctcatt cggggtctgg gccatggagc 481ctttggggag gtgtatgaag gccaggtgtc cggaatgccc aacgacccaa gccccctgca 541agtggctgtg aagacgctgc ctgaagtgtg ctctgaacag gacgaactgg atttcctcat 601ggaagccctg atcatcagca aattcaacca ccagaacatt gttcgctgca ttggggtgag 661cctgcaatcc ctgccccggt tcatcctgct ggagctcatg gcggggggag acctcaagtc 721cttcctccga gagacccgcc ctcgcccgag ccagccctcc tccctggcca tgctggacct 781tctgcacgtg gctcgggaca ttgcctgtgg ctgtcagtat ttggaggaaa accacttcat 841ccaccgagac attgctgcca gaaactgcct cttgacctgt ccaggccctg gaagagtggc 901caagattgga gacttcggga tggcccgaga catctacagg gcgagctact atagaaaggg 961aggctgtgcc atgctgccag ttaagtggat gcccccagag gccttcatgg aaggaatatt 1021cacttctaaa acagacacat ggtcctttgg agtgctgcta tgggaaatct tttctcttgg 1081atatatgcca taccccagca aaagcaacca ggaagttctg gagtttgtca ccagtggagg 1141ccggatggac ccacccaaga actgccctgg gcctgtatac cggataatga ctcagtgctg 1201gcaacatcag cctgaagaca ggcccaactt tgccatcatt ttggagagga ttgaatactg 1261cacccaggac ccggatgtaa tcaacaccgc tttgccgata gaatatggtc cacttgtgga 1321agaggaagag aaagtgcctg tgaggcccaa ggaccctgag ggggttcctc ctctcctggt 1381ctctcaacag gcaaaacggg aggaggagcg cagcccagct gccccaccac ctctgcctac 1441cacctcctct ggcaaggctg caaagaaacc cacagctgca gaggtctctg ttcgagtccc 1501tagagggccg gccgtggaag ggggacacgt gaatatggca ttctctcagt ccaaccctcc 1561ttcggagttg cacagggtcc acggatccag aaacaagccc accagcttgt ggaacccaac 1621gtacggctcc tggtttacag agaaacccac caaaaagaat aatcctatag caaagaagga 1681gccacacgag aggggtaacc tggggctgga gggaagctgt actgtcccac ctaacgttgc 1741aactgggaga cttccggggg cctcactgct cctagagccc tcttcgctga ctgccaatat 1801gaaggaggta cctctgttca ggctacgtca cttcccttgt gggaatgtca attacggcta 1861ccagcaacag ggcttgccct tagaagccgc tactgcccct ggagctggtc attacgagga 1921taccattctg aaaagcaaga atagcatgaa ccagcctggg ccctgagctc ggtcgcacac 1981tcacttctct tccttgggat ccctaagacc gtgg
EML4-ALK sudden change 5a protein sequence (BAG75148.1；GI:209837706)
1mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva
61svkksvsskv yrrkhqelqa mqmelqspey klsklrtsti mtdynpnycf agktssisdl
121kevprknitl irglghgafg evyegqvsgm pndpsplqva vktlpevcse qdeldflmea 181liiskfnhqn ivrcigvslq slprfillel maggdlksfl retrprpsqp sslamldllh 241vardiacgcq yleenhfihr diaarncllt cpgpgrvaki gdfgmardiy rasyyrkggc 301amlpvkwmpp eafmegifts ktdtwsfgvl lweifslgym pypsksnqev lefvtsggrm 361dppkncpgpv yrimtqcwqh qpedrpnfai ilerieyctq dpdvintalp ieygplveee 421ekvpvrpkdp egvppllvsq qakreeersp aappplptts sgkaakkpta aevsvrvprg 481pavegghvnm afsqsnppse lhrvhgsrnk ptslwnptyg swftekptkk nnpiakkeph 541ergnlglegs ctvppnvatg rlpgasllle pssltanmke vplfrlrhfp cgnvnygyqq 601qglpleaata pgaghyedti lksknsmnqp gp
EML4-ALK sudden change 5b protein sequence (AB374365.1；GI:209837707)
1actctgtcgg tccgctgaat gaagtgcccg cccctctaag cccggagccc ggcgctttcc
61ccgcaagatg gacggtttcg ccggcagtct cgatgatagt atttctgctg caagtacttc
121tgatgttcaa gatcgcctgt cagctcttga gtcacgagtt cagcaacaag aagatgaaat 181cactgtgcta aaggcggctt tggctgatgt tttgaggcgt cttgcaatct ctgaagatca 241tgtggcctca gtgaaaaaat cagtctcaag taaaggttca gagctcaggg gaggatatgg 301agatccaggg aggcttcctg taggaagtgg cctgtgtagt gcttcaaggg ccaggctgcc 361aggccatgtt gcagctgacc acccacctgc agtgtaccgc cggaagcacc aggagctgca 421agccatgcag atggagctgc agagccctga gtacaagctg agcaagctcc gcacctcgac 481catcatgacc gactacaacc ccaactactg ctttgctggc aagacctcct ccatcagtga 541cctgaaggag gtgccgcgga aaaacatcac cctcattcgg ggtctgggcc atggagcctt 601tggggaggtg tatgaaggcc aggtgtccgg aatgcccaac gacccaagcc ccctgcaagt 661ggctgtgaag acgctgcctg aagtgtgctc tgaacaggac gaactggatt tcctcatgga 721agccctgatc atcagcaaat tcaaccacca gaacattgtt cgctgcattg gggtgagcct 781gcaatccctg ccccggttca tcctgctgga gctcatggcg gggggagacc tcaagtcctt 841cctccgagag acccgccctc gcccgagcca gccctcctcc ctggccatgc tggaccttct 901gcacgtggct cgggacattg cctgtggctg tcagtatttg gaggaaaacc acttcatcca 961ccgagacatt gctgccagaa actgcctctt gacctgtcca ggccctggaa gagtggccaa 1021gattggagac ttcgggatgg cccgagacat ctacagggcg agctactata gaaagggagg 1081ctgtgccatg ctgccagtta agtggatgcc cccagaggcc ttcatggaag gaatattcac 1141ttctaaaaca gacacatggt cctttggagt gctgctatgg gaaatctttt ctcttggata 1201tatgccatac cccagcaaaa gcaaccagga agttctggag tttgtcacca gtggaggccg 1261gatggaccca cccaagaact gccctgggcc tgtataccgg ataatgactc agtgctggca 1321acatcagcct gaagacaggc ccaactttgc catcattttg gagaggattg aatactgcac 1381ccaggacccg gatgtaatca acaccgcttt gccgatagaa tatggtccac ttgtggaaga 1441ggaagagaaa gtgcctgtga ggcccaagga ccctgagggg gttcctcctc tcctggtctc 1501tcaacaggca aaacgggagg aggagcgcag cccagctgcc ccaccacctc tgcctaccac 1561ctcctctggc aaggctgcaa agaaacccac agctgcagag gtctctgttc gagtccctag 1621agggccggcc gtggaagggg gacacgtgaa tatggcattc tctcagtcca accctccttc 1681ggagttgcac agggtccacg gatccagaaa caagcccacc agcttgtgga acccaacgta 1741cggctcctgg tttacagaga aacccaccaa aaagaataat cctatagcaa agaaggagcc 1801acacgagagg ggtaacctgg ggctggaggg aagctgtact gtcccaccta acgttgcaac 1861tgggagactt ccgggggcct cactgctcct agagccctct tcgctgactg ccaatatgaa 1921ggaggtacct ctgttcaggc tacgtcactt cccttgtggg aatgtcaatt acggctacca 1981gcaacagggc ttgcccttag aagccgctac tgcccctgga gctggtcatt acgaggatac 2041cattctgaaa agcaagaata gcatgaacca gcctgggccc tgagctcggt cgcacactca 2101cttctcttcc ttgggatccc taagaccgtg g
EML4-ALK sudden change 5b protein sequence (BAG75149.1；GI:209837708)
1mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva
61svkksvsskg selrggygdp grlpvgsglc sasrarlpgh vaadhppavy rrkhqelqam
121qmelqspeyk lsklrtstim tdynpnycfa gktssisdlk evprknitli rglghgafge 181vyegqvsgmp ndpsplqvav ktlpevcseq deldflmeal iiskfnhqni vrcigvslqs 241lprfillelm aggdlksflr etrprpsqps slamldllhv ardiacgcqy leenhfihrd 301iaarnclltc pgpgrvakig dfgmardiyr asyyrkggca mlpvkwmppe afmegiftsk 361tdtwsfgvll weifslgymp ypsksnqevl efvtsggrmd ppkncpgpvy rimtqcwqhq 421pedrpnfaii lerieyctqd pdvintalpi eygplveeee kvpvrpkdpe gvppllvsqq 481akreeerspa appplpttss gkaakkptaa evsvrvprgp avegghvnma fsqsnppsel 541hrvhgsrnkp tslwnptygs wftekptkkn npiakkephe rgnlglegsc tvppnvatgr 601lpgaslllep ssltanmkev plfrlrhfpc gnvnygyqqq glpleaatap gaghyedtil 661ksknsmnqpg p
EML4-ALK suddenly change 6 nucleotide sequences (AB462411.1；GI:227452648)
1tactctgtcg gtccgctgaa tgaagtgccc gcccctctaa gcccggagcc cggcgctttc
61cccgcaagat ggacggtttc gccggcagtc tcgatgatag tatttctgct gcaagtactt
121ctgatgttca agatcgcctg tcagctcttg agtcacgagt tcagcaacaa gaagatgaaa 181tcactgtgct aaaggcggct ttggctgatg ttttgaggcg tcttgcaatc tctgaagatc 241atgtggcctc agtgaaaaaa tcagtctcaa gtaaaggcca accaagccct cgagcagtta 301ttcccatgtc ctgtataacc aatggaagtg gtgcaaacag aaaaccaagt cataccagtg 361ctgtctcaat tgcaggaaaa gaaactcttt catctgctgc taaaagtggt acagaaaaaa 421agaaagaaaa accacaagga cagagagaaa aaaaagagga atctcattct aatgatcaaa 481gtccacaaat tcgagcatca ccttctcccc agccctcttc acaacctctc caaatacaca 541gacaaactcc agaaagcaag aatgctactc ccaccaaaag cataaaacga ccatcaccag 601ctgaaaagtc acataattct tgggaaaatt cagatgatag ccgtaataaa ttgtcgaaaa 661taccttcaac acccaaatta ataccaaaag ttaccaaaac tgcagacaag cataaagatg 721tcatcatcaa ccaagaagga gaatatatta aaatgtttat gcgcggtcgg ccaattacca 781tgttcattcc ttccgatgtt gacaactatg atgacatcag aacggaactg cctcctgaga 841agctcaaact ggagtgggca tatggttatc gaggaaagga ctgtagagct aatgtttacc 901ttcttccgac cggggaaata gtttatttca ttgcatcagt agtagtacta tttaattatg 961aggagagaac tcagcgacac tacctgggcc atacagactg tgtgaaatgc cttgctatac 1021atcctgacaa aattaggatt gcaactggac agatagctgg cgtggataaa gatggaaggc 1081ctctacaacc ccacgtcaga gtgtgggatt ctgttactct atccacactg cagattattg 1141gacttggcac ttttgagcgt ggagtaggat gcctggattt ttcaaaagca gattcaggtg 1201ttcatttatg tgttattgat gactccaatg agcatatgct tactgtatgg gactggcaga 1261ggaaagcaaa aggagcagaa ataaagacaa caaatgaagt tgttttggct gtggagtttc 1321acccaacaga tgcaaatacc ataattacat gcggtaaatc tcatattttc ttctggacct 1381ggagcggcaa ttcactaaca agaaaacagg gaatttttgg gaaatatgaa aagccaaaat 1441ttgtgcagtg tttagcattc ttggggaatg gagatgttct tactggagac tcaggtggag 1501tcatgcttat atggagcaaa actactgtag agcccacacc tgggaaagga cctaaaggaa 1561gtggcctgtg tagtgcttca agggccaggc tgccaggcca tgttgcagct gaccacccac 1621ctgcagtgta ccgccggaag caccaggagc tgcaagccat gcagatggag ctgcagagcc 1681ctgagtacaa gctgagcaag ctccgcacct cgaccatcat gaccgactac aaccccaact 1741actgctttgc tggcaagacc tcctccatca gtgacctgaa ggaggtgccg cggaaaaaca 1801tcaccctcat tcggggtctg ggccatggag cctttgggga ggtgtatgaa ggccaggtgt 1861ccggaatgcc caacgaccca agccccctgc aagtggctgt gaagacgctg cctgaagtgt 1921gctctgaaca ggacgaactg gatttcctca tggaagccct gatcatcagc aaattcaacc 1981accagaacat tgttcgctgc attggggtga gcctgcaatc cctgccccgg ttcatcctgc 2041tggagctcat ggcgggggga gacctcaagt ccttcctccg agagacccgc cctcgcccga 2101gccagccctc ctccctggcc atgctggacc ttctgcacgt ggctcgggac attgcctgtg 2161gctgtcagta tttggaggaa aaccacttca tccaccgaga cattgctgcc agaaactgcc 2221tcttgacctg tccaggccct ggaagagtgg ccaagattgg agacttcggg atggcccgag 2281acatctacag ggcgagctac tatagaaagg gaggctgtgc catgctgcca gttaagtgga 2341tgcccccaga ggccttcatg gaaggaatat tcacttctaa aacagacaca tggtcctttg 2401gagtgctgct atgggaaatc ttttctcttg gatatatgcc ataccccagc aaaagcaacc 2461aggaagttct ggagtttgtc accagtggag gccggatgga cccacccaag aactgccctg 2521ggcctgtata ccggataatg actcagtgct ggcaacatca gcctgaagac aggcccaact 2581ttgccatcat tttggagagg attgaatact gcacccagga cccggatgta atcaacaccg 2641ctttgccgat agaatatggt ccacttgtgg aagaggaaga gaaagtgcct gtgaggccca 2701aggaccctga gggggttcct cctctcctgg tctctcaaca ggcaaaacgg gaggaggagc 2761gcagcccagc tgccccacca cctctgccta ccacctcctc tggcaaggct gcaaagaaac 2821ccacagctgc agaggtctct gttcgagtcc ctagagggcc ggccgtggaa gggggacacg 2881tgaatatggc attctctcag tccaaccctc cttcggagtt gcacagggtc cacggatcca 2941gaaacaagcc caccagcttg tggaacccaa cgtacggctc ctggtttaca gagaaaccca 3001ccaaaaagaa taatcctata gcaaagaagg agccacacga gaggggtaac ctggggctgg 3061agggaagctg tactgtccca cctaacgttg caactgggag acttccgggg gcctcactgc 3121tcctagagcc ctcttcgctg actgccaata tgaaggaggt acctctgttc aggctacgtc 3181acttcccttg tgggaatgtc aattacggct accagcaaca gggcttgccc ttagaagccg 3241ctactgcccc tggagctggt cattacgagg ataccattct gaaaagcaag aatagcatga 3301accagcctgg gccctgagct cggtcgcaca ctcacttctc ttccttggga tccctaagac 3361cgtgg
EML4-ALK suddenly change 6 protein sequences (BAH57335.1；GI:227452649)
1mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva
61svkksvsskg qpspravipm scitngsgan rkpshtsavs iagketlssa aksgtekkke
121kpqgqrekke eshsndqspq iraspspqps sqplqihrqt pesknatptk sikrpspaek 181shnswensdd srnklskips tpklipkvtk tadkhkdvii nqegeyikmf mrgrpitmfi 241psdvdnyddi rtelppeklk lewaygyrgk dcranvyllp tgeivyfias vvvlfnyeer 301tqrhylghtd cvkclaihpd kiriatgqia gvdkdgrplq phvrvwdsvt lstlqiiglg 361tfergvgcld fskadsgvhl cviddsnehm ltvwdwqrka kgaeikttne vvlavefhpt 421dantiitcgk shiffwtwsg nsltrkqgif gkyekpkfvq claflgngdv ltgdsggvml 481iwskttvept pgkgpkgsgl csasrarlpg hvaadhppav yrrkhqelqa mqmelqspey 541klsklrtsti mtdynpnycf agktssisdl kevprknitl irglghgafg evyegqvsgm 601pndpsplqva vktlpevcse qdeldflmea liiskfnhqn ivrcigvslq slprfillel 661maggdlksfl retrprpsqp sslamldllh vardiacgcq yleenhfihr diaarncllt 721cpgpgrvaki gdfgmardiy rasyyrkggc amlpvkwmpp eafmegifts ktdtwsfgvl 781lweifslgym pypsksnqev lefvtsggrm dppkncpgpv yrimtqcwqh qpedrpnfai 841ilerieyctq dpdvintalp ieygplveee ekvpvrpkdp egvppllvsq qakreeersp 901aappplptts sgkaakkpta aevsvrvprg pavegghvnm afsqsnppse lhrvhgsrnk 961ptslwnptyg swftekptkk nnpiakkeph ergnlglegs ctvppnvatg rlpgasllle 1021pssltanmke vplfrlrhfp cgnvnygyqq qglpleaata pgaghyedti lksknsmnqp 1081gp
EML4-ALK suddenly change 7 nucleotide sequences (AB462412.1；GI:227452650)
1tactctgtcg gtccgctgaa tgaagtgccc gcccctctaa gcccggagcc cggcgctttc
61cccgcaagat ggacggtttc gccggcagtc tcgatgatag tatttctgct gcaagtactt
121ctgatgttca agatcgcctg tcagctcttg agtcacgagt tcagcaacaa gaagatgaaa 181tcactgtgct aaaggcggct ttggctgatg ttttgaggcg tcttgcaatc tctgaagatc 241atgtggcctc agtgaaaaaa tcagtctcaa gtaaaggcca accaagccct cgagcagtta 301ttcccatgtc ctgtataacc aatggaagtg gtgcaaacag aaaaccaagt cataccagtg 361ctgtctcaat tgcaggaaaa gaaactcttt catctgctgc taaaagtggt acagaaaaaa 421agaaagaaaa accacaagga cagagagaaa aaaaagagga atctcattct aatgatcaaa 481gtccacaaat tcgagcatca ccttctcccc agccctcttc acaacctctc caaatacaca 541gacaaactcc agaaagcaag aatgctactc ccaccaaaag cataaaacga ccatcaccag 601ctgaaaagtc acataattct tgggaaaatt cagatgatag ccgtaataaa ttgtcgaaaa 661taccttcaac acccaaatta ataccaaaag ttaccaaaac tgcagacaag cataaagatg 721tcatcatcaa ccaagaagga gaatatatta aaatgtttat gcgcggtcgg ccaattacca 781tgttcattcc ttccgatgtt gacaactatg atgacatcag aacggaactg cctcctgaga 841agctcaaact ggagtgggca tatggttatc gaggaaagga ctgtagagct aatgtttacc 901ttcttccgac cggggaaata gtttatttca ttgcatcagt agtagtacta tttaattatg 961aggagagaac tcagcgacac tacctgggcc atacagactg tgtgaaatgc cttgctatac 1021atcctgacaa aattaggatt gcaactggac agatagctgg cgtggataaa gatggaaggc 1081ctctacaacc ccacgtcaga gtgtgggatt ctgttactct atccacactg cagattattg 1141gacttggcac ttttgagcgt ggagtaggat gcctggattt ttcaaaagca gattcaggtg 1201ttcatttatg tgttattgat gactccaatg agcatatgct tactgtatgg gactggcaga 1261ggaaagcaaa aggagcagaa ataaagacaa caaatgaagt tgttttggct gtggagtttc 1321acccaacaga tgcaaatacc ataattacat gcggtaaatc tcatattttc ttctggacct 1381ggagcggcaa ttcactaaca agaaaacagg gaatttttgg gaaatatgaa aagccaaaat 1441ttgtgcagtg tttagcattc ttggggaatg gagatgttct tactggagac tcaggtggag 1501tcatgcttat atggagcaaa actactgtag agcccacacc tgggaaagga cctaaaggtg 1561tatatcaaat cagcaaacaa atcaaagctc atgatggcag tgtgttcaca ctttgtcaga 1621tgagaaatgg gatgttatta actggaggag ggaaagacag aaaaataatt ctgtgggatc 1681atgatctgaa tcctgaaaga gaaatagagc accaggagct gcaagccatg cagatggagc 1741tgcagagccc tgagtacaag ctgagcaagc tccgcacctc gaccatcatg accgactaca 1801accccaacta ctgctttgct ggcaagacct cctccatcag tgacctgaag gaggtgccgc 1861ggaaaaacat caccctcatt cggggtctgg gccatggagc ctttggggag gtgtatgaag 1921gccaggtgtc cggaatgccc aacgacccaa gccccctgca agtggctgtg aagacgctgc 1981ctgaagtgtg ctctgaacag gacgaactgg atttcctcat ggaagccctg atcatcagca 2041aattcaacca ccagaacatt gttcgctgca ttggggtgag cctgcaatcc ctgccccggt 2101tcatcctgct ggagctcatg gcggggggag acctcaagtc cttcctccga gagacccgcc 2161ctcgcccgag ccagccctcc tccctggcca tgctggacct tctgcacgtg gctcgggaca 2221ttgcctgtgg ctgtcagtat ttggaggaaa accacttcat ccaccgagac attgctgcca 2281gaaactgcct cttgacctgt ccaggccctg gaagagtggc caagattgga gacttcggga 2341tggcccgaga catctacagg gcgagctact atagaaaggg aggctgtgcc atgctgccag 2401ttaagtggat gcccccagag gccttcatgg aaggaatatt cacttctaaa acagacacat 2461ggtcctttgg agtgctgcta tgggaaatct tttctcttgg atatatgcca taccccagca 2521aaagcaacca ggaagttctg gagtttgtca ccagtggagg ccggatggac ccacccaaga 2581actgccctgg gcctgtatac cggataatga ctcagtgctg gcaacatcag cctgaagaca 2641ggcccaactt tgccatcatt ttggagagga ttgaatactg cacccaggac ccggatgtaa 2701tcaacaccgc tttgccgata gaatatggtc cacttgtgga agaggaagag aaagtgcctg 2761tgaggcccaa ggaccctgag ggggttcctc ctctcctggt ctctcaacag gcaaaacggg 2821aggaggagcg cagcccagct gccccaccac ctctgcctac cacctcctct ggcaaggctg 2881caaagaaacc cacagctgca gaggtctctg ttcgagtccc tagagggccg gccgtggaag 2941ggggacacgt gaatatggca ttctctcagt ccaaccctcc ttcggagttg cacaaggtcc 3001acggatccag aaacaagccc accagcttgt ggaacccaac gtacggctcc tggtttacag 3061agaaacccac caaaaagaat aatcctatag caaagaagga gccacacgac aggggtaacc 3121tggggctgga gggaagctgt actgtcccac ctaacgttgc aactgggaga cttccggggg 3181cctcactgct cctagagccc tcttcgctga ctgccaatat gaaggaggta cctctgttca 3241ggctacgtca cttcccttgt gggaatgtca attacggcta ccagcaacag ggcttgccct 3301tagaagccgc tactgcccct ggagctggtc attacgagga taccattctg aaaagcaaga 3361atagcatgaa ccagcctggg ccctgagctc ggtcgcacac tcacttctct tccttgggat 3421ccctaagacc gtgga
EML4-ALK suddenly change 7 protein sequences (BAH57336.1；GI:227452651)
1mdgfagsldd sisaastsdv qdrlsalesr vqqqedeitv lkaaladvlr rlaisedhva
61svkksvsskg qpspravipm scitngsgan rkpshtsavs iagketlssa aksgtekkke
121kpqgqrekke eshsndqspq iraspspqps sqplqihrqt pesknatptk sikrpspaek 181shnswensdd srnklskips tpklipkvtk tadkhkdvii nqegeyikmf mrgrpitmfi 241psdvdnyddi rtelppeklk lewaygyrgk dcranvyllp tgeivyfias vvvlfnyeer 301tqrhylghtd cvkclaihpd kiriatgqia gvdkdgrplq phvrvwdsvt lstlqiiglg 361tfergvgcld fskadsgvhl cviddsnehm ltvwdwqrka kgaeikttne vvlavefhpt 421dantiitcgk shiffwtwsg nsltrkqgif gkyekpkfvq claflgngdv ltgdsggvml 481iwskttvept pgkgpkgvyq iskqikahdg svftlcqmrn gmlltgggkd rkiilwdhdl 541npereiehqe lqamqmelqs peyklsklrt stimtdynpn ycfagktssi sdlkevprkn 601itlirglghg afgevyegqv sgmpndpspl qvavktlpev cseqdeldfl mealiiskfn 661hqnivrcigv slqslprfil lelmaggdlk sflretrprp sqpsslamld llhvardiac 721gcqyleenhf ihrdiaarnc lltcpgpgrv akigdfgmar diyrasyyrk ggcamlpvkw 781mppeafmegi ftsktdtwsf gvllweifsl gympypsksn qevlefvtsg grmdppkncp 841gpvyrimtqc wqhqpedrpn faiileriey ctqdpdvint alpieygplv eeeekvpvrp 901kdpegvppll vsqqakreee rspaappplp ttssgkaakk ptaaevsvrv prgpaveggh 961vnmafsqsnp pselhkvhgs rnkptslwnp tygswftekp tkknnpiakk ephdrgnlgl 1021egsctvppnv atgrlpgasl llepssltan mkevplfrlr hfpcgnvnyg yqqqglplea 1081atapgaghye dtilksknsm nqpgp
KIF5B-ALK nucleotide sequence (AB462413.1；GI:227452652)
1tgcgagaaag atggcggacc tggccgagtg caacatcaaa gtgatgtgtc gcttcagacc
61tctcaacgag tctgaagtga accgcggcga caagtacatc gccaagtttc agggagaaga
121cacggtcgtg atcgcgtcca agccttatgc atttgatcgg gtgttccagt caagcacatc 181tcaagagcaa gtgtataatg actgtgcaaa gaagattgtt aaagatgtac ttgaaggata 241taatggaaca atatttgcat atggacaaac atcctctggg aagacacaca caatggaggg 301taaacttcat gatccagaag gcatgggaat tattccaaga atagtgcaag atatttttaa 361ttatatttac tccatggatg aaaatttgga atttcatatt aaggtttcat attttgaaat 421atatttggat aagataaggg acctgttaga tgtttcaaag accaaccttt cagttcatga 481agacaaaaac cgagttccct atgtaaaggg gtgcacagag cgttttgtat gtagtccaga 541tgaagttatg gataccatag atgaaggaaa atccaacaga catgtagcag ttacaaatat 601gaatgaacat agctctagga gtcacagtat atttcttatt aatgtcaaac aagagaacac 661acaaacggaa caaaagctga gtggaaaact ttatctggtt gatttagctg gtagtgaaaa 721ggttagtaaa actggagctg aaggtgctgt gctggatgaa gctaaaaaca tcaacaagtc 781actttctgct cttggaaatg ttatttctgc tttggctgag ggtagtacat atgttccata 841tcgagatagt aaaatgacaa gaatccttca agattcatta ggtggcaact gtagaaccac 901tattgtaatt tgctgctctc catcatcata caatgagtct gaaacaaaat ctacactctt 961atttggccaa agggccaaaa caattaagaa cacagtttgt gtcaatgtgg agttaactgc 1021agaacagtgg aaaaagaagt atgaaaaaga aaaagaaaaa aataagatcc tgcggaacac 1081tattcagtgg cttgaaaatg agctcaacag atggcgtaat ggggagacgg tgcctattga 1141tgaacagttt gacaaagaga aagccaactt ggaagctttc acagtggata aagatattac 1201tcttaccaat gataaaccag caaccgcaat tggagttata ggaaatttta ctgatgctga 1261aagaagaaag tgtgaagaag aaattgctaa attatacaaa cagcttgatg acaaggatga 1321agaaattaac cagcaaagtc aactggtaga gaaactgaag acgcaaatgt tggatcagga 1381ggagcttttg gcatctacca gaagggatca agacaatatg caagctgagc tgaatcgcct 1441tcaagcagaa aatgatgcct ctaaagaaga agtgaaagaa gttttacagg ccctagaaga 1501acttgctgtc aattatgatc agaagtctca ggaagttgaa gacaaaacta aggaatatga 1561attgcttagt gatgaattga atcagaaatc ggcaacttta gcgagtatag atgctgagct 1621tcagaaactt aaggaaatga ccaaccacca gaaaaaacga gcagctgaga tgatggcatc 1681tttactaaaa gaccttgcag aaataggaat tgctgtggga aataatgatg taaagcagcc 1741tgagggaact ggcatgatag atgaagagtt cactgttgca agactctaca ttagcaaaat 1801gaagtcagaa gtaaaaacca tggtgaaacg ttgcaagcag ttagaaagca cacaaactga 1861gagcaacaaa aaaatggaag aaaatgaaaa ggagttagca gcatgtcagc ttcgtatctc 1921tcaacatgaa gccaaaatca agtcattgac tgaatacctt caaaatgtgg aacaaaagaa 1981aagacagttg gaggaatctg tcgatgccct cagtgaagaa ctagtccagc ttcgagcaca 2041agagaaagtc catgaaatgg aaaaggagca cttaaataag gttcagactg caaatgaagt 2101taagcaagct gttgaacagc agatccagag ccatagagaa actcatcaaa aacagatcag 2161tagtttgaga gatgaagtag aagcaaaagc aaaacttatt actgatcttc aagaccaaaa 2221ccagaaaatg atgttagagc aggaacgtct aagagtagaa catgagaagt tgaaagccac 2281agatcaggaa aagagcagaa aactacatga acttacggtt atgcaagata gacgagaaca 2341agcaagacaa gacttgaagg gtttggaaga gacagtggca aaagaacttc agactttaca 2401caacctgcgc aaactctttg ttcaggacct ggctacaaga gttaaaaaga gtgctgagat 2461tgattctgat gacaccggag gcagcgctgc tcagaagcaa aaaatctcct ttcttgaaaa 2521taatcttgaa cagctcacta aagtgcacaa acagttggta cgtgataatg cagatctccg 2581ctgtgaactt cctaagttgg aaaagcgact tcgagctaca gctgagagag tgaaagcttt 2641ggaatcagca ctgaaagaag ctaaagaaaa tgcatctcgt gatcgcaaac gctatcagca 2701agaagtagat cgcataaagg aagcagtcag gtcaaagaat atggccagaa gagggcattc 2761tgcacagatt gtgtaccgcc ggaagcacca ggagctgcaa gccatgcaga tggagctgca 2821gagccctgag tacaagctga gcaagctccg cacctcgacc atcatgaccg actacaaccc 2881caactactgc tttgctggca agacctcctc catcagtgac ctgaaggagg tgccgcggaa 2941aaacatcacc ctcattcggg gtctgggcca tggcgccttt ggggaggtgt atgaaggcca 3001ggtgtccgga atgcccaacg acccaagccc cctgcaagtg gctgtgaaga cgctgcctga 3061agtgtgctct gaacaggacg aactggattt cctcatggaa gccctgatca tcagcaaatt 3121caaccaccag aacattgttc gctgcattgg ggtgagcctg caatccctgc cccggttcat 3181cctgctggag ctcatggcgg ggggagacct caagtccttc ctccgagaga cccgccctcg 3241cccgagccag ccctcctccc tggccatgct ggaccttctg cacgtggctc gggacattgc 3301ctgtggctgt cagtatttgg aggaaaacca cttcatccac cgagacattg ctgccagaaa 3361ctgcctcttg acctgtccag gccctggaag agtggccaag attggagact tcgggatggc 3421ccgagacatc tacagggcga gctactatag aaagggaggc tgtgccatgc tgccagttaa 3481gtggatgccc ccagaggcct tcatggaagg aatattcact tctaaaacag acacatggtc 3541ctttggagtg ctgctatggg aaatcttttc tcttggatat atgccatacc ccagcaaaag 3601caaccaggaa gttctggagt ttgtcaccag tggaggccgg atggacccac ccaagaactg 3661ccctgggcct gtataccgga taatgactca gtgctggcaa catcagcctg aagacaggcc 3721caactttgcc atcattttgg agaggattga atactgcacc caggacccgg atgtaatcaa 3781caccgctttg ccgatagaat atggtccact tgtggaagag gaagagaaag tgcctgtgag 3841gcccaaggac cctgaggggg ttcctcctct cctggtctct caacaggcaa aacgggagga 3901ggagcgcagc ccagctgccc caccacctct gcctaccacc tcctctggca aggctgcaaa 3961gaaacccaca gctgcagagg tctctgttcg agtccctaga gggccggccg tggaaggggg 4021acacgtgaat atggcattct ctcagtccaa ccctccttcg gagttgcaca aggtccacgg 4081atccagaaac aagcccacca gcttgtggaa cccaacgtac ggctcctggt ttacagagaa 4141acccaccaaa aagaataatc ctatagcaaa gaaggagcca cacgacaggg gtaacctggg 4201gctggaggga agctgtactg tcccacctaa cgttgcaact gggagacttc cgggggcctc 4261actgctccta gagccctctt cgctgactgc caatatgaag gaggtacctc tgttcaggct 4321acgtcacttc ccttgtggga atgtcaatta cggctaccag caacagggct tgcccttaga 4381agccgctact gcccctggag ctggtcatta cgaggatacc attctgaaaa gcaagaatag 4441catgaaccag cctgggccct gagctcggtc gcacactca
KIF5B-ALK protein sequence (BAH57337.1；GI:227452653)
1madlaecnik vmcrfrplne sevnrgdkyi akfqgedtvv iaskpyafdr vfqsstsqeq
61vyndcakkiv kdvlegyngt ifaygqtssg kthtmegklh dpegmgiipr ivqdifnyiy
121smdenlefhi kvsyfeiyld kirdlldvsk tnlsvhedkn rvpyvkgcte rfvcspdevm 181dtidegksnr hvavtnmneh ssrshsifli nvkqentqte qklsgklylv dlagsekvsk 241tgaegavlde akninkslsa lgnvisalae gstyvpyrds kmtrilqdsl ggncrttivi 301ccspssynes etkstllfgq raktikntvc vnveltaeqw kkkyekekek nkilrntiqw 361lenelnrwrn getvpideqf dkekanleaf tvdkditltn dkpataigvi gnftdaerrk 421ceeeiaklyk qlddkdeein qqsqlveklk tqmldqeell astrrdqdnm qaelnrlqae 481ndaskeevke vlqaleelav nydqksqeve dktkeyells delnqksatl asidaelqkl 541kemtnhqkkr aaemmasllk dlaeigiavg nndvkqpegt gmideeftva rlyiskmkse 601vktmvkrckq lestqtesnk kmeenekela acqlrisqhe akikslteyl qnveqkkrql 661eesvdalsee lvqlraqekv hemekehlnk vqtanevkqa veqqiqshre thqkqisslr 721deveakakli tdlqdqnqkm mleqerlrve heklkatdqe ksrklheltv mqdrreqarq 781dlkgleetva kelqtlhnlr klfvqdlatr vkksaeidsd dtggsaaqkq kisflennle 841qltkvhkqlv rdnadlrcel pklekrlrat aervkalesa lkeakenasr drkryqqevd 901rikeavrskn marrghsaqi vyrrkhqelq amqmelqspe yklsklrtst imtdynpnyc 961fagktssisd lkevprknit lirglghgaf gevyegqvsg mpndpsplqv avktlpevcs 1021eqdeldflme aliiskfnhq nivrcigvsl qslprfille lmaggdlksf lretrprpsq 1081psslamldll hvardiacgc qyleenhfih rdiaarncll tcpgpgrvak igdfgmardi 1141yrasyyrkgg camlpvkwmp peafmegift sktdtwsfgv llweifslgy mpypsksnqe 1201vlefvtsggr mdppkncpgp vyrimtqcwq hqpedrpnfa iilerieyct qdpdvintal 1261pieygplvee eekvpvrpkd pegvppllvs qqakreeers paappplptt ssgkaakkpt 1321aaevsvrvpr gpavegghvn mafsqsnpps elhkvhgsrn kptslwnpty gswftekptk 1381knnpiakkep hdrgnlgleg sctvppnvat grlpgaslll epssltanmk evplfrlrhf 1441pcgnvnygyq qqglpleaat apgaghyedt ilksknsmnq pgp
NPM-ALK sequence (t (2;5)(p23;Q35 chromosome translocation) *
TPM3-ALK sequence (t (1;2)(p25;P23) chromosome translocation) *
TFGXL-ALK nucleotide sequence (AF390893.1；GI:20269389)
1atgaacggac agttggatct aagtgggaag ctaatcatca aagctcaact tggggaggat
61attcggcgaa ttcctattca taatgaagat attacttatg atgaattagt gctaatgatg
121caacgagttt tcagaggaaa acttctgagt aatgatgaag taacaataaa gtataaagat 181gaagatggag atcttataac aatttttgat agttctgacc tttcctttgc aattcagtgc 241agtaggatac tgaaactgac attatttgtt aatggccagc caagacccct tgaatcaagt 301caggtgaaat atctccgtcg agaactgata gaacttcgaa ataaagtgaa tcgtttattg 361gatagcttgg aaccacctgg agaaccagga ccttccacca atattcctga aaatgatact 421gtggatggta gggaagaaaa gtctgcttct gattcttctg gaaaacagtc tactcaggtt 481atggcagcaa gtatgtctgc ttttgatcct ttaaaaaacc aagatgaaat caataaaaat 541gttatgtcag cgtttggctt aacagatgat caggtttcag ggccacccag tgctcctgca 601gaagatcgtt caggaacacc cgacagcatt gcttcctcct cctcagcagc tcacccacca 661ggcgttcagc cacagcagcc accatataca ggagctcaga ctcaagcagg tcagattgaa 721gtgtaccgcc ggaagcacca ggagctgcaa gccatgcaga tggagctgca gagccctgag 781tacaagctga gcaagctccg cacctcgacc atcatgaccg actacaaccc caactactgc 841tttgctggca agacctcctc catcagtgac ctgaaggagg tgccgcggaa aaacatcacc 901ctcattcggg gtctgggcca tggcgccttt ggggaggtgt atgaaggcca ggtgtccgga 961atgcccaacg acccaagccc cctgcaagtg gctgtgaaga cgctgcctga agtgtgctct 1021gaacaggacg aactggattt cctcatggaa gccctgatca tcagcaaatt caaccaccag 1081aacattgttc gctgcattgg ggtgagcctg caatccctgc cccggttcat cctgctggag 1141ctcatggcgg ggggagacct caagtccttc ctccgagaga cccgccctcg cccgagccag 1201ccctcctccc tggccatgct ggaccttctg cacgtggctc gggacattgc ctgtggctgt 1261cagtatttgg aggaaaacca cttcatccac cgagacattg ctgccagaaa ctgcctcttg 1321acctgtccag gccctggaag agtggccaag attggagact tcgggatggc ccgagacatc 1381tacagggcga gctactatag aaagggaggc tgtgccatgc tgccagttaa gtggatgccc 1441ccagaggcct tcatggaagg aatattcact tctaaaacag acacatggtc ctttggagtg 1501ctgctatggg aaatcttttc tcttggatat atgccatacc ccagcaaaag caaccaggaa 1561gttctggagt ttgtcaccag tggaggccgg atggacccac ccaagaactg ccctgggcct 1621gtataccgga taatgactca gtgctggcaa catcagcctg aagacaggcc caactttgcc 1681atcattttgg agaggattga atactgcacc caggacccgg atgtaatcaa caccgctttg 1741ccgatagaat atggtccact tgtggaagag gaagagaaag tgcctgtgag gcccaaggac 1801cctgaggggg ttcctcctct cctggtctct caacaggcaa aacgggagga ggagcgcagc 1861ccagctgccc caccacctct gcctaccacc tcctctggca aggctgcaaa gaaacccaca 1921gctgcagagg tctctgttcg agtccctaga gggccggccg tggaaggggg acacgtgaat 1981atggcattct ctcagtccaa ccctccttcg gagttgcaca aggtccacgg atccagaaac 2041aagcccacca gcttgtggaa cccaacgtac ggctcctggt ttacagagaa acccaccaaa 2101aagaataatc ctatagcaaa gaaggagcca cacgacaggg gtaacctggg gctggaggga 2161agctgtactg tcccacctaa cgttgcaact gggagacttc cgggggcctc actgctccta 2221gagccctctt cgctgactgc caatatgaag gaggtacctc tgttcaggct acgtcacttc 2281ccttgtggga atgtcaatta cggctaccag caacagggct tgcccttaga agccgctact 2341gcccctggag ctggtcatta cgaggatacc attctgaaaa gcaagaatag catgaaccag 2401cctgggccct ga
TFGXL-ALK protein sequence (AAM17922.1；GI:20269390) *
1mngqldlsgk liikaqlged irripihned itydelvlmm qrvfrgklls ndevtikykd
61edgdlitifd ssdlsfaiqc srilkltlfv ngqprpless qvkylrreli elrnkvnrll
121dsleppgepg pstnipendt vdgreeksas dssgkqstqv maasmsafdp lknqdeinkn 181vmsafgltdd qvsgppsapa edrsgtpdsi assssaahpp gvqpqqppyt gaqtqagqie 241vyrrkhqelq amqmelqspe yklsklrtst imtdynpnyc fagktssisd lkevprknit 301lirglghgaf gevyegqvsg mpndpsplqv avktlpevcs eqdeldflme aliiskfnhq 361nivrcigvsl qslprfille lmaggdlksf lretrprpsq psslamldll hvardiacgc 421qyleenhfih rdiaarncll tcpgpgrvak igdfgmardi yrasyyrkgg camlpvkwmp 481peafmegift sktdtwsfgv llweifslgy mpypsksnqe vlefvtsggr mdppkncpgp 541vyrimtqcwq hqpedrpnfa iilerieyct qdpdvintal pieygplvee eekvpvrpkd 601pegvppllvs qqakreeers paappplptt ssgkaakkpt aaevsvrvpr gpavegghvn 661mafsqsnpps elhkvhgsrn kptslwnpty gswftekptk knnpiakkep hdrgnlgleg 721sctvppnvat grlpgaslll epssltanmk evplfrlrhf pcgnvnygyq qqglpleaat 781apgaghyedt ilksknsmnq pgp
TFGL-ALK nucleotide sequence(AF143407.1；GI:6739534)
1cctccgcaag ccgtctttct ctagagttgt atatatagaa catcctggag tccaccatga
61acggacagtt ggatctaagt gggaagctaa tcatcaaagc tcaacttggg gaggatattc
121ggcgaattcc tattcataat gaagatatta cttatgatga attagtgcta atgatgcaac 181gagttttcag aggaaaactt ctgagtaatg atgaagtaac aataaagtat aaagatgaag 241atggagatct tataacaatt tttgatagtt ctgacctttc ctttgcaatt cagtgcagta 301ggatactgaa actgacatta tttgttaatg gccagccaag accccttgaa tcaagtcagg 361tgaaatatct ccgtcgagaa ctgatagaac ttcgaaataa agtgaatcgt ttattggata 421gcttggaacc acctggagaa ccaggacctt ccaccaatat tcctgaaaat gatactgtgg 481atggtaggga agaaaagtct gcttctgatt cttctggaaa acagtctact caggttatgg 541cagcaagtat gtctgctttt gatcctttaa aaaaccaaga tgaaatcaat aaaaatgtta 601tgtcagcgtt tggcttaaca gatgatcagg tttcagtgta ccgccggaag caccaggagc 661tgcaagccat gcagatggag ctgcagagcc ctgagtacaa gctgagcaag ctccgcacct 721cgaccatcat gaccgactac aaccccaact actgctttgc tggcaagacc tcctccatca 781gtgacctgaa ggaggtgccg cggaaaaaca tcaccctcat tcggggtctg ggccatggcg 841cctttgggga ggtgtatgaa ggccaggtgt ccggaatgcc caacgaccca agccccctgc 901aagtggctgt gaagacgctg cctgaagtgt gctctgaaca ggacgaactg gatttcctca 961tggaagccct gatcatcagc aaattcaacc accagaacat tgttcgctgc attggggtga 1021gcctgcaatc cctgccccgg ttcatcctgc tggagctcat ggcgggggga gacctcaagt 1081ccttcctccg agagacccgc cctcgcccga gccagccctc ctccctggcc atgctggacc 1141ttctgcacgt ggctcgggac attgcctgtg gctgtcagta tttggaggaa aaccacttca 1201tccaccgaga cattgctgcc agaaactgcc tcttgacctg tccaggccct ggaagagtgg 1261ccaagattgg agacttcggg atggcccgag acatctacag ggcgagctac tatagaaagg 1321gaggctgtgc catgctgcca gttaagtgga tgcccccaga ggccttcatg gaaggaatat 1381tcacttctaa aacagacaca tggtcctttg gagtgctgct atgggaaatc ttttctcttg 1441gatatatgcc ataccccagc aaaagcaacc aggaagttct ggagtttgtc accagtggag 1501gccggatgga cccacccaag aactgccctg ggcctgtata ccggataatg actcagtgct 1561ggcaacatca gcctgaagac aggcccaact ttgccatcat tttggagagg attgaatact 1621gcacccagga cccggatgta atcaacaccg ctttgccgat agaatatggt ccacttgtgg 1681aagaggaaga gaaagtgcct gtgaggccca aggaccctga gggggttcct cctctcctgg 1741tctctcaaca ggcaaaacgg gaggaggagc gcagcccagc tgccccacca cctctgccta 1801ccacctcctc tggcaaggct gcaaagaaac ccacagctgc agaggtctct gttcgagtcc 1861ctagagggcc ggccgtggaa gggggacacg tgaatatggc attctctcag tccaaccctc 1921cttcggagtt gcacaaggtc cacggatcca gaaacaagcc caccagcttg tggaacccaa 1981cgtacggctc ctggtttaca gagaaaccca ccaaaaagaa taatcctata gcaaagaagg 2041agccacacga caggggtaac ctggggctgg agggaagctg tactgtccca cctaacgttg 2101caactgggag acttccgggg gcctcactgc tcctagagcc ctcttcgctg actgccaata 2161tgaaggaggt acctctgttc aggctacgtc acttcccttg tgggaatgtc aattacggct 2221accagcaaca gggcttgccc ttagaagccg ctactgcccc tggagctggt cattacgagg 2281ataccattct gaaaagcaag aatagcatga accagcctgg gccctgagct cggtcgcaca 2341ctcacttctc ttccttggga tccctaagac cgtggaggag agagaggcaa tggctccttc 2401acaaaccaga gaccaaatgt cacgttttgt tttgtgccaa cctattttga agtaccacca 2461aaaaagctgt attttgaaaa tgctttagaa aggttttgag catgggttca tcctattctt 2521tcgaaagaag aaaatatcat aaaaatgagt gataaataca aggcccagat gtggttgcat 2581aaggttttta tgcatgtttg ttgtatactt ccttatgctt cttttaaatt gtgtgtgctc 2641tgcttcaatg tagtcagaat tagctgcttc tatgtttcat agttggggtc atagatgttt 2701ccttgccttg ttgatgtgga catgagccat ttgaggggag agggaacgga aataaaggag 2761ttatttgtaa tgactaaaa
TFGL-ALK protein sequence (AAF27292.1；GI:6739535) *
1mngqldlsgk liikaqlged irripihned itydelvlmm qrvfrgklls ndevtikykd
61edgdlitifd ssdlsfaiqc srilkltlfv ngqprpless qvkylrreli elrnkvnrll 121dsleppgepg pstnipendt vdgreeksas dssgkqstqv maasmsafdp lknqdeinkn 181vmsafgltdd qvsvyrrkhq elqamqmelq speyklsklr tstimtdynp nycfagktss 241isdlkevprk nitlirglgh gafgevyegq vsgmpndpsp lqvavktlpe vcseqdeldf 301lmealiiskf nhqnivrcig vslqslprfi llelmaggdl ksflretrpr psqpsslaml 361dllhvardia cgcqyleenh fihrdiaarn clltcpgpgr vakigdfgma rdiyrasyyr 421kggcamlpvk wmppeafmeg iftsktdtws fgvllweifs lgympypsks nqevlefvts 481ggrmdppknc pgpvyrimtq cwqhqpedrp nfaiilerie yctqdpdvin talpieygpl 541veeeekvpvr pkdpegvppl lvsqqakree erspaapppl pttssgkaak kptaaevsvr 601vprgpavegg hvnmafsqsn ppselhkvhg srnkptslwn ptygswftek ptkknnpiak 661kephdrgnlg legsctvppn vatgrlpgas lllepsslta nmkevplfrl rhfpcgnvny 721gyqqqglple aatapgaghy edtilkskns mnqpgp
TFGS-ALK nucleotide sequence (AF125093.1；GI:7229260)
1cctccgcaag ccgtctttct ctagagttgt atatatagaa catcctggag tccaccatga
61acggacagtt ggatctaagt gggaagctaa tcatcaaagc tcaacttggg gaggatattc 121ggcgaattcc tattcataat gaagatatta cttatgatga attagtgcta atgatgcaac 181gagttttcag aggaaaactt ctgagtaatg atgaagtaac aataaagtat aaagatgaag 241atggagatct tataacaatt tttgatagtt ctgacctttc ctttgcaatt cagtgcagta 301ggatactgaa actgacatta tttgttaatg gccagccaag accccttgaa tcaagtcagg 361tgaaatatct ccgtcgagaa ctgatagaac ttcgaaataa agtgaatcgt ttattggata 421gcttggaacc acctggagaa ccaggacctt ccaccaatat tcctgaaaat gtgtaccgcc 481ggaagcacca ggagctgcaa gccatgcaga tggagctgca gagccctgag tacaagctga 541gcaagctccg cacctcgacc atcatgaccg actacaaccc caactactgc tttgctggca 601agacctcctc catcagtgac ctgaaggagg tgccgcggaa aaacatcacc ctcattcggg 661gtctgggcca tggcgccttt ggggaggtgt atgaaggcca ggtgtccgga atgcccaacg 721acccaagccc cctgcaagtg gctgtgaaga cgctgcctga agtgtgctct gaacaggacg 781aactggattt cctcatggaa gccctgatca tcagcaaatt caaccaccag aacattgttc 841gctgcattgg ggtgagcctg caatccctgc cccggttcat cctgctggag ctcatggcgg 901ggggagacct caagtccttc ctccgagaga cccgccctcg cccgagccag ccctcctccc 961tggccatgct ggaccttctg cacgtggctc gggacattgc ctgtggctgt cagtatttgg 1021aggaaaacca cttcatccac cgagacattg ctgccagaaa ctgcctcttg acctgtccag 1081gccctggaag agtggccaag attggagact tcgggatggc ccgagacatc tacagggcga 1141gctactatag aaagggaggc tgtgccatgc tgccagttaa gtggatgccc ccagaggcct 1201tcatggaagg aatattcact tctaaaacag acacatggtc ctttggagtg ctgctatggg 1261aaatcttttc tcttggatat atgccatacc ccagcaaaag caaccaggaa gttctggagt 1321ttgtcaccag tggaggccgg atggacccac ccaagaactg ccctgggcct gtataccgga 1381taatgactca gtgctggcaa catcagcctg aagacaggcc caactttgcc atcattttgg 1441agaggattga atactgcacc caggacccgg atgtaatcaa caccgctttg ccgatagaat 1501atggtccact tgtggaagag gaagagaaag tgcctgtgag gcccaaggac cctgaggggg 1561ttcctcctct cctggtctct caacaggcaa aacgggagga ggagcgcagc ccagctgccc 1621caccacctct gcctaccacc tcctctggca aggctgcaaa gaaacccaca gctgcagagg 1681tctctgttcg agtccctaga gggccggccg tggaaggggg acacgtgaat atggcattct 1741ctcagtccaa ccctccttcg gagttgcaca aggtccacgg atccagaaac aagcccacca 1801gcttgtggaa cccaacgtac ggctcctggt ttacagagaa acccaccaaa aagaataatc 1861ctatagcaaa gaaggagcca cacgacaggg gtaacctggg gctggaggga agctgtactg 1921tcccacctaa cgttgcaact gggagacttc cgggggcctc actgctccta gagccctctt 1981cgctgactgc caatatgaag gaggtacctc tgttcaggct acgtcacttc ccttgtggga 2041atgtcaatta cggctaccag caacagggct tgcccttaga agccgctact gcccctggag 2101ctggtcatta cgaggatacc attctgaaaa gcaagaatag catgaaccag cctgggccct 2161gagctcggtc gcacactcac ttctcttcct tgggatccct aagaccgtgg aggagagaga 2221ggcaatggct ccttcacaaa ccagagacca aatgtcacgt tttgttttgt gccaacctat 2281tttgaagtac caccaaaaaa gctgtatttt gaaaatgctt tagaaaggtt ttgagcatgg 2341gttcatccta ttctttcgaa agaagaaaat atcataaaaa tgagtgataa atacaaggcc 2401cagatgtggt tgcataaggt ttttatgcat gtttgttgta tacttcctta tgcttctttt 2461aaattgtgtg tgctctgctt caatgtagtc agaattagct gcttctatgt ttcatagttg 2521gggtcataga tgtttccttg ccttgttgat gtggacatga gccatttgag gggagaggga 2581acggaaataa aggagttatt tgtaatgact aaaa
TFGS-ALK protein sequence (AAF42734.1；GI:7229261) *
1mngqldlsgk liikaqlged irripihned itydelvlmm qrvfrgklls ndevtikykd
61edgdlitifd ssdlsfaiqc srilkltlfv ngqprpless qvkylrreli elrnkvnrll
121dsleppgepg pstnipenvy rrkhqelqam qmelqspeyk lsklrtstim tdynpnycfa 181gktssisdlk evprknitli rglghgafge vyegqvsgmp ndpsplqvav ktlpevcseq 241deldflmeal iiskfnhqni vrcigvslqs lprfillelm aggdlksflr etrprpsqps 301slamldllhv ardiacgcqy leenhfihrd iaarnclltc pgpgrvakig dfgmardiyr 361asyyrkggca mlpvkwmppe afmegiftsk tdtwsfgvll weifslgymp ypsksnqevl 421efvtsggrmd ppkncpgpvy rimtqcwqhq pedrpnfaii lerieyctqd pdvintalpi 481eygplveeee kvpvrpkdpe gvppllvsqq akreeerspa appplpttss gkaakkptaa 541evsvrvprgp avegghvnma fsqsnppsel hkvhgsrnkp tslwnptygs wftekptkkn 601npiakkephd rgnlglegsc tvppnvatgr lpgaslllep ssltanmkev plfrlrhfpc 661gnvnygyqqq glpleaatap gaghyedtil ksknsmnqpg p
ATIC-ALK sequence (inv (2) (p23;Q35) chromosome translocation) *
CLTC-ALK sequence (t (2;17)(p23;Q23) chromosome translocation) *
MSN-ALK nucleotide sequence (AF295356.1；GI:14625823)
1aactccgctg cctttgccgc caccatgccc aaaacgatca gtgtgcgtgt gaccaccatg
61gatgcagagc tggagtttgc catccagccc aacaccaccg ggaagcagct atttgaccag
121gtggtgaaaa ctattggctt gagggaagtt tggttctttg gtctgcagta ccaggacact 181aaaggtttct ccacctggct gaaactcaat aagaaggtga ctgcccagga tgtgcggaag 241gaaagccccc tgctctttaa gttccgtgcc aagttctacc ctgaggatgt gtccgaggaa 301ttgattcagg acatcactca gcgcctgttc tttctgcaag tgaaagaggg cattctcaat 361gatgatattt actgcccgcc tgagaccgct gtgctgctgg cctcgtatgc tgtccagtct 421aagtatggcg acttcaataa ggaagtgcat aagtctggct acctggccgg agacaagttg 481ctcccgcaga gagtcctgga acagcacaaa ctcaacaagg accagtggga ggagcggatc 541caggtgtggc atgaggaaca ccgtggcatg ctcagggagg atgctgtcct ggaatatctg 601aagattgctc aagatctgga gatgtatggt gtgaactact tcagcatcaa gaacaagaaa 661ggctcagagc tgtggctggg ggtggatgcc ctgggtctca acatctatga gcagaatgac 721agactaactc ccaagatagg cttcccctgg agtgaaatca ggaacatctc tttcaatgat 781aagaaatttg tcatcaagcc cattgacaaa aaagccccgg acttcgtctt ctatgctccc 841cggctgcgga ttaacaagcg gatcttggcc ttgtgcatgg ggaaccatga actatacatg 901cgccgtcgca agcctgatac cattgaggtg cagcagatga aggcacaggc ccgggaggag 961aagcaccaga agcagatgga gcgtgctatg ctggaaaatg agaagaagaa gcgtgaaatg 1021gcagagaagg agaaagagaa gattgaacgg gagaaggagg agctgatgga gaggctgaag 1081cagatcgagg aacagactaa gaaggctcag caagaactgg aagaacagac ccgtagggct 1141ctggaacttg agcaggaacg gaagcgtgcc cagagcgagg ctgaaaagct ggccaaggag 1201cgtcaagaag ctgaagaggc caaggaggcc ttgctgcagg cctcccggga ccagaaaaag 1261actcaggaac agctggcctt ggaaatggca gagctgacag ctcgaatctc ccagctggag 1321atggcccgac agaagaagga gagtgaggct gtggagtggc agcagaagca ggagctgcaa 1381gccatgcaga tggagctgca gagccctgag tacaagctga gcaagctccg cacctcgacc 1441atcatgaccg actacaaccc caactactgc tttgctggca agacctcctc catcagtgac 1501ctgaaggagg tgccgcggaa aaacatcacc ctcattcggg gtctgggcca tggcgccttt 1561ggggaggtgt atgaaggcca ggtgtccgga atgcccaacg acccaag
MSN-ALK protein sequence (AAK71522.1；GI:14625824) *
1mpktisvrvt tmdaelefai qpnttgkqlf dqvvktiglr evwffglqyq dtkgfstwlk
61lnkkvtaqdv rkespllfkf rakfypedvs eeliqditqr lfflqvkegi lnddiycppe
121tavllasyav qskygdfnke vhksgylagd kllpqrvleq hklnkdqwee riqvwheehr 181gmlredavle ylkiaqdlem ygvnyfsikn kkgselwlgv dalglniyeq ndrltpkigf 241pwseirnisf ndkkfvikpi dkkapdfvfy aprlrinkri lalcmgnhel ymrrrkpdti 301evqqmkaqar eekhqkqmer amlenekkkr emaekekeki erekeelmer lkqieeqtkk 361aqqeleeqtr raleleqerk raqseaekla kerqeaeeak eallqasrdq kktqeqlale 421maeltarisq lemarqkkes eavewqqkqe lqamqmelqs peyklsklrt stimtdynpn 481ycfagktssi sdlkevprkn itlirglghg afgevyegqv sgmpndp
TPM4-ALK little coding mutation sequence (AF362887.1；GI:14010353)
1cgagaagttg agggagaaag gcgggcccgg gaacaggctg aggctgaggt ggcctccttg
61aaccgtagga tccagctggt tgaagaagag ctggaccgtg ctcaggagcg tgcggaggtg 121tctgaactaa aatgtggtga cctggaagaa gaactcaaga atgttactaa caatctgaaa 181tctctggagg ctgcatctga aaagtattct gaaaaggagg acaaatatga agaagaaatt 241aaacttctgt ctgacaaact gaaagaggct gagacccgtg ctgaatttgc agagagaacg 301gttgcaaaac tggaaaagac aattgatgac ctggaagtgt acctccggaa gcaccaagag 361ctgcaagcca tgcagatgga gctgcagagc cctgagtaca agctgagcaa gctccgcacc 421ctcgac
TPM4-ALK little protein mutant sequence (AAK51964.1；GI:14010354)
1revegerrar eqaeaevasl nrriqlveee ldraqeraev selkcgdlee elknvtnnlk
61sleaasekys ekedkyeeei kllsdklkea etraefaert vaklektidd levylrkhqe 121lqamqmelqs peyklsklrt ld
TPM4-ALK big coding mutation sequence (AF362886.1；GI:14010351)
1ctggcagagt cccgttgccg agagatggat gagcagatta gactgatgga ccagaacctg
61aagtgtctga gtgctgctga agaaaagtac tctcaaaaag aagataaata tgaggaagaa 121atcaagattc ttactgataa actcaaggag gcagagaccc gtgctgaatt tgcagagaga 181acggttgcaa aactggaaaa gacaattgat gacctggaag tgtaccgccg gaagcaccag 241gagctgcaag ccatgcagat ggagctgcag agccctgagt acaagctgag caagctccgc 301acctcgac
TPM4-ALK larger protein mutant nucleotide sequence (AAK51963.1；GI:14010352)
1laesrcremd eqirlmdqnl kclsaaeeky sqkedkyeee ikiltdklke aetraefaer
61tvaklektid dlevyrrkhq elqamqmelq speyklsklr tst
MYH9-ALK sequence (t (2；22)(p23；Q11.2) chromosome translocation) *
RANBP2-ALK sequence (t (2；2)(p23；Or inv (2) (p23 q13);Q11-13) chromosome translocation) *
ALO17-ALK sequence (t (2;17)(p23;Q25) chromosome translocation) *
CARS-ALK sequence (t (2;11;2)(p23;p15;Q31) chromosome translocation) *
In addition to MSN-ALK and MYH-9, all fused proteins contain last 563 aminoacid of ALK.MSN-ALK Last 567 and 566 aminoacid is contained respectively with MYH9.
" ALK sudden change " is usually directed to a kind of nucleotide in a relevant anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase sequence and/or aminoacid sequence Change.But in certain embodiments, " ALK sudden change " relate to specific anaplastic lymphoma kinase ALK Alk receptor tyrosine kinase ALK inhibitor (as PF-02341066 and/or PDD) produced suddenly change in advance when processing.Such as, in wild type alk protein (NP_004295) At 1156, at cysteine (C1156) and/or 1196, leucine (L1196) sports a kind of different aminoacid respectively, they As described herein ALK inhibitor is produced resistance.In one embodiment, C1156 position include a tyrosine and/or L1196 position includes a methionine.Those of ordinary skill in the art it can also be seen that with C1156 in wild type alk protein and Amino acid position quantity the most corresponding for L1196 is different with other correlated series (such as ALK homolog, ALK fusion protein etc.), no The sudden change value in advance of its response ALK inhibitor (such as PF-02341066 and/or PDD) can be affected.The ordinary skill people of this area Member is it can also be seen that determine should be able to be according to genetic code known between the aminoacid sequence of a specific protein and nucleotide sequence (not shown) is definitely for protein coding.Equally, the nucleotide sequence of a specific nucleotide and aminoacid sequence Determine known between that correspondence also can encode according to the nucleotide that genetic code determines.
Genetic code
One important and known feature of genetic code is that it is too much, therefore to use most aminoacid to prepare protein, It is accomplished by using more than one coding nucleotide triplet (the most as shown in the figure).Therefore, some different nucleotide sequences May be used for encoding a specific aminoacid sequence.These nucleotide sequences are considered as that function is identical, because they can be in institute Have in organism and produce identical aminoacid sequence (although some biological physical ability more efficiently translates some sequences than them).This Outward, in a specific aminoacid sequence, a methylated purine or pyrimidine mutant are found sometimes.This methylate not The encoding relation between trinucleotide codons and corresponding aminoacid can be affected.In addition those of ordinary skill in the art knows How one specific codon nucleotide suddenlys change, thus explicitly changes a coded amino acid alkali in associated cryptographic sublist Base.Such as, the codon of Cys-1156 is " TGC ", and Tyr is probably " TAT " or " TAC ".Therefore, by position 2 The codon G at place replaces with A just can encoding tyrosine rather than cysteine.Those of ordinary skill in the art can be carried out Similar operation designs other sudden changes.
" binding compounds " refers to a kind of binding compounds, such as a kind of little molecule, a kind of antibody, a kind of peptide, a kind of peptide or non-peptide Part, a kind of protein, a kind of oligonucleotide, a kind of oligonucleotide analogs, such as a kind of peptide nucleotide, a kind of agglutinin or Any other molecular entity, they can be combined with a target protein or molecule, or can be with a kind of analyte, as one is multiple Foreign protein combines and forms stable complex.
" in conjunction with base " refers to the molecule that any molecular marker can connect directly or indirectly, and it specifically can be combined with a kind of analyte. Include that in conjunction with base molecular weight is about up to 1000 daltonian antibody without limitation, antibodies compound, peptide, protein, Nucleic acid and organic molecule, the atom that it contains includes hydrogen, carbon, oxygen, nitrogen, sulfur and phosphorus.
One " biomarker "or" labelling " it is the gene that can change, mRNA or protein, wherein said change and cancer Disease is relevant.Described change can be that tumor tissues or tumor cell be normal with one or health tissues or cell (such as a control) And quantity, structure and/or the activity when suffering from cancer compare, its quantity, structure and/or the change of activity.The such as present invention A described labelling with related to cancer or a labelling of premature ACK anti-cancer therapies can be at tumor tissues or tumor cells In have a nucleotide sequence different from a normal healthy tissues or cell, aminoacid sequence, chromosome translocation, interior Chromosome inversion, copy number, expression, protein level, protein active or methylation state.Additionally, " a mark Note " includes the molecule of a structural change, such as sudden change (comprising a sudden change), such as different from wild-type sequence nucleoside Acid or amino acid levels, such as, be replaced in the tissue suffering from the diseases such as cancer or cell, delete or insert.
Term " cancer " or " tumor " refer to the cell with typical carcinogenic cells, such as its diffusion uncontrolled, the most extremely, turn Move potential, fast-growth and the rate of increase high, some feature of cell morphological characteristic.Cancerous cell is typically a tumor, but this Cell can be independently present in an animal, it is also possible to is a kind of non-tumorigenesis tumor cell, such as a kind of leukaemia.This In invention, term " cancer " includes precancerous lesion and malignant tumor.Cancer includes B cell tumor without limitation, as multiple Myeloma, this special macroglobulinemia of Walden, heavy chain disease, such as α chain is sick, gamma chain disease, and mu chain is sick, benign monoclonal third Plant gammopathy, immunocytic amyloidosis, melanoma, breast carcinoma, pulmonary carcinoma (such as nonsmall-cell lung cancer or NSCLC)), Bronchogenic carcinoma, colorectal cancer, carcinoma of prostate, cancer of pancreas, gastric cancer, ovarian cancer, bladder cancer, brain or central nerve neuroma, Peripheral nervous system neoplasms, the esophageal carcinoma, cervical cancer, uterus or carcinoma of endometrium, oral cavity or laryngocarcinoma, hepatocarcinoma, renal carcinoma, testis Ball cancer, cancer of bile ducts, small intestinal or vermiform appendix cancer, salivary-gland carcinoma, thyroid carcinoma, adrenal carcinoma, osteosarcoma, chondrosarcoma, blood Tissue cancer, adenocarcinoma, inflammation myofibroblastoma, gastrointestinal stromal tumor (GIST), colorectal carcinoma, multiple myeloma (MM), myelodysplastic syndrome (MDS), myeloproliferative disease (MPD), acute lymphoblastic leukemia (ALL), Acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), true property is red Cytosis, Hodgkin lymphoma, non-Hodgkin lymphoma (NHL), soft tissue sarcoma, fibrosarcoma, myxosarcoma, Liposarcoma, osteosarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, Mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, Sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatocarcinoma, cancer of bile ducts, Choriocarcinoma, spermocytoma, embryonal carcinoma, nephroblastoma, bladder cancer, epithelial cancer, glioma, astrocytoma, Medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, brain Film tumor, neuroblastoma, retinoblastoma, follicular lymphoma, diffusivity large B cell lymphoid tumor, jacket cell drenches Bar tumor, hepatocarcinoma, thyroid carcinoma, gastric cancer, incidence cancer, small cell carcinoma, primary thrombocytosis, the bone of unknown cause Myeloid tissue heteroplasia disease, leukemia chronic eosinophilic, systemic mastocytosis, similar eosinophilia disease, Chronic eosinophilic leukemia, neuroendocrine tumor, benign tumor etc..
" chemotherapeutics " refers to a kind of chemical substance, and such as a kind of cytotoxicity or cytostatic reagent, it can be one It is used for treating when planting situation such as cancer.
" complementary " refers to the most general of the complementary between two nucleotide sequence chains or between two identical nucleotide sequence chains Read.It is known that an adenine residue in first nucleotide sequence can second nucleoside the most antiparallel with A residue in acid sequence forms specific hydrogen bond (" base pairing "), and residue therein is thymus pyrimidine or uracil. It is also known that a cytosine residues in first nucleotide chain can second core the most antiparallel with one A residue in nucleotide sequence forms specific base pairing, and residue therein is guanine.One the first sequence of one nucleic acid Row and second complementary of same or different nucleotide, if the two sequence is antiparallel, and first In sequence, at least a nucleotide residue can be with a residue base pairing in the second sequence.In certain embodiments, institute The First ray stated includes a Part I, and described second area includes a Part II, i.e. when the first and second parts It is antiparallel, then in Part I, the nucleotide residue of the most about 50%, 75%, 90% or 95 can be with second The nucleotide residue base pairing divided.In other embodiments, all nucleotide residues in Part I can be with second Nucleotide residue base pairing in Fen.
Described " copy number of gene " or " copy number of labelling " refer to DNA sequence encode in a cell one specific The quantity of gene prod.A specific gene in a usual mammal has two copies.But described copy number can To be increased by gene amplification or duplication, or reduced by deletion.
One labelling is " fixing " on substrate, if it is to be connected by covalent bond or non-covalent bond with substrate, then substrate Just can divide from substrate without by substantial amounts of labelling with a kind of liquid rinse (such as standard citrate, pH value 7.4) Separate out.
" danger coefficient " of the present invention refers to use a kind of statistical method to obtain a kind of assessment to risk." dangerous system Number " it is the ratio organized with another of one group of anticipated risk.Such as will employ patient that a kind of ALK inhibitor carries out treating and do not make With comparing of a kind of ALK inhibitor, determine whether ALK inhibitor can increase the interval time of palindromia effectively, especially It is to suddenly change for ALK.The most as described herein, use those tumor tissues of ALK inhibitor for treating have produced ALK The object of sudden change, its effect can be more worse than the effect not producing ALK sudden change.
" ALK suppresses reagent " of the present invention or " ALK inhibitor " refer to that one can suppress the bioactive compound of ALK. Biological activity also includes the patient reaction listed by the application.Typical ALK suppression reagent includes PF-02341066 without limitation, PDD, 2-methyl isophthalic acid 1-(2-methyl-propyl)-4-oxygen-4,5,6,11,12,13-hexahydro-2H-indazole [5,4-α] pyrroles [3,4-c] carbazole-8-base [4-(dimethylamino) benzyl] methyl carbamate, (1S, 2S, 3R, 4R)-3-({ 5-chloro-2-[(1- Ethyl-2,3,4,5-tetrahydrochysene-6-methoxyl group-2-oxygen-1H-1-benzazepine-7-base) amino]-4-pyrimidine } amino) dicyclo [2.2.1] hept-5-alkene-2-Methanamide and NVP-TAE684(see for example PNAS104:270-275,2007;Choi,Y.L. et al.(2008)Cancer Res.68:4971-2976;And Biochemistry48:3600-3609,2009, this Bright incorporated by reference).
Term " homology " or " homology " can exchange use, and they refer to two polynucleotide sequences or two peptide sequences Between similar sequences there is tightened up tetraploid rice.Term " homology or homology percentage rate " and " homology or homology % " Refer to the percentage rate of similar sequences in two or more polynucleotide sequences or two or more peptide sequence sequences. " sequence similarity " refers to that the similar percentage rate of base-pair sequence in two or more polynucleotide sequence is (with arbitrarily closing Suitable method determines).Two or more sequences can have similarity or the global similarity of 0-100% anywhere. Homology or similarity can be obtained by the same position of relatively each sequence.When being phase on a position in comparative sequences Same nucleotide or aminoacid, then these molecules are exactly homology on this position.The likelihood or same of polynucleotide sequence Source rate refers to that these polynucleotide sequences have nucleotide homology or a quantity function of coupling on position.One of peptide sequence Homology refers to that these peptide sequences have a quantity function of position upper amino acid homology.One homology of peptide sequence or Likelihood refers to that these peptide sequences have a quantity function of position upper amino acid.Term of the present invention " essence homology " Refer at least 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more homology.
" suppression " of cancer, if at least a cancer symptoms is alleviated, terminates, delays or stop.As used in the present invention , if can reduce, slow down, slow down or prophylaxis of cancer recurs or transfer, then cancer also " is suppressed ".
One " labeling nucleic acid " or " biomarker nucleic acid " is that one is by a label coding of the present invention or corresponding therewith Nucleic acid (such as DNA, mRNA, cDNA).The most this marker nucleic acid molecule includes DNA(such as, genomic DNA and cDNA), This DNA includes any nucleotide sequence listed by table 1 or the part in these sequences, or the complementary series of these sequences or Hybridization sequences.This marker nucleic acid molecule also includes RNA, and this RNA includes any nucleotide sequence listed by table 1 or these sequences A part in row, or the complementary series of these sequences, the most all of breast pyrimidine residue is replaced by uracil residues.A kind of " labelled protein " is the albumen by a label coding of the present invention or the albumen of correspondence.A kind of labelled protein includes Arbitrary sequence listed by table 1 or the whole protein sequence of sequence fragment coding or partial-length proteins.Term " albumen " and " many Peptide " in the present invention connotation identical.
" normally " expression of " normally " copy number of one labelling or a labelling refers in a biological specimen, as From a destination object not suffering from cancer, as human body gathers containing sputum, bronchoalveolar lavage, hydrothorax, group Knitting, whole blood, serum, blood plasma, oral cavity is scraped, saliva, cerebrospinal fluid, urine, in the sample of feces and bone marrow, described labelling Expression and copy number.
ALK gene sudden change and/or the one " overexpression " or " the highest of gene outcome (labelling as listed in table 1) Expression, copy number and/or activity " be in a detection sample, a kind of expression, copy number and/or specific activity The expression of standard or copy number detection mistake are bigger, and it can be at least one and control sample (as do not suffered from cancer from one Health objects in gather sample) in ALK gene sudden change and/or gene outcome (labelling as listed in table 1) expression or ALK gene sudden change and/or base in two, three, four, five or ten or higher multiples of copy number, or the most several control sample Average expression level or two, three, four, five or ten or higher multiples of copy number because of product (labelling as listed in table 1).
Term " probe " refers to any molecule that can be optionally combined with a specific target molecules, the most of the present invention A labelling.Probe both can be synthesized by the those of ordinary skill of the present invention, it is also possible to is prepared by suitable biological preparation.For Detection target molecule, can make probe especially with labelling as described in the present invention.The Typical molecular that can serve as probe is non- Restrictively include ribonucleic acid, DNA (deoxyribonucleic acid), protein, antibody and organic monomer.
" RECIST " is an abbreviation, means " evaluation criteria of solid tumor reaction ", and the standard that it is announced is when cancer is suffered from Sb.'s illness took a favorable turn over the course for the treatment of for person (" reaction "), keeps constant (" stablizing ") or deteriorates (" progress ").Such as National Cancer research magazine 92 phase No. 3 on February 2nd, 2000 has issued for the reaction determined according to RECIST standard, RECIST Standard can include that other similar definition and rule are arranged.Those of ordinary skill in the art understands that the present invention can use The definition that RECIST standard determines, such as " PR ", " CR ", " SD " and " PD ".
" reaction " and its other tense that " response " of the present invention treats refer to that an object uses a kind of ALK suppression The reaction produced after agent.In one embodiment, an object can produce reaction, i.e. this object after using a kind of ALK inhibitor In tumor can the most slowly increase by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more. In another embodiment, an object can produce reaction after using a kind of ALK inhibitor, i.e. uses suitably detection, such as matter Amount or volume etc. can find that the tumor in this object about reduces 5%, 10%, 20%, 30%, 40%, 50% or more. In another embodiment, reaction, i.e. its average life can be produced after an a kind of ALK inhibitor of object use can not accept by ratio The average life for the treatment of increases about 5%, and 10%, 20%, 30%, 40%, 50% or more.In another embodiment, One object can produce reaction, i.e. this object after using a kind of ALK inhibitor a DFS increased, and entirety is deposited Motility rate or increase deterioration times.Several method can be used to determine whether patient responds treatment, and these methods include above-mentioned RECIST standard.
" sample ", " tissue samples ", " patient's sample ", " patient's cell or tissue sample " or " specimen " each mean from one One tissue of object or patient gathers the similar cellular of acquisition.The source of this tissue samples is probably from one fresh, cold Freeze and/or preserve the solid tissue obtained in organ, tissue samples, section, or aspirate；Blood or any blood constituent；Body Liquid such as cerebrospinal fluid, amniotic fluid, ascites or tissue fluid；Or in objective body, produce or grow the cell of random time.Described Tissue samples can be containing the non-natural compound that mixes with natural tissues, such as preservative, and anticoagulant, buffer agent, fixative, Nutrient, antibiotic etc..
If more measured than the standard error of evaluation quantity by one, or at least evaluation quantity two, three, four, five, ten Or the amount of more the most times makes the amount of this labelling more higher or lower than normal level respectively, then the amount of a labelling, such as ALK Gene mutation or the expression of gene outcome (labelling as listed in table 1) or copy number, in an object be " substantially " high In or less than the normal amount of a labelling.Or the amount of this labelling is considered " substantially " and is higher or lower than in subject Normal amount, if this amount is at least the two of labelling normal amount, three, four, five times, higher or lower times.
In the present invention, " critical event " refers to one event when patient is ill, and those of ordinary skill in the art thinks that it is weight Want.Typical critical event includes ID without limitation, dead, and recurrence determines that the pathological changes of patient shifts, Patient disease recurrence or patient disease from an any of the above described stage development to another stage.One critical event can be by this The those of ordinary skill in field uses OS, TTP and/or RECIST or other reaction normal to assess any critical event determined.
In the present invention, " object " and " patient " can exchange use.Term " object " used in the present invention and " right As " refer to animal, as include non-primate (such as cattle, pig, horse, donkey, sheep, camel, cat, Canis familiaris L., Cavia porcellus, greatly Mus, mice, sheep) and primate (such as monkey, such as machin, gorilla, chimpanzee and the mankind) interior mammal.
" time course " used in the present invention refers to primary event and time quantum between event subsequently.Such as one patient Cancer, its time course can relate to the disease of a patient and notable thing of measuring can be used in this lysis to survey Amount, the most such as first event can be to be diagnosed, and event below can be transfer.
" development time " or " TTP " refers to start to development or a kind of cancer or a measurement time of inspection from treatment.Check May be from a research to terminate or from the change treated.Development time can also be expressed as a kind of probability, such as one In individual assessment event development point, development time can represent its probability of Free Development within a specific time, and the time is Refer to that treatment started to the time between development or inspection. One " polynucleotide are transcribed " is polynucleotide (such as one RNA, a cDNA or RNA or cDNA Homology), its all or part of complementary or homology with a mature rna, wherein mature rna is by the one of the present invention Post processing (such as splicing) is transcribed and transcribed normally to labelling, if any, the reverse transcription transcribed and transcribe obtains.
" treating ", other form of " process " and this word limits a kind of cancer all referring to use ALK inhibitor Growth, makes a kind of cancer reduce weight or volume, extends anticipated time-to-live or the development time etc. of tumor of object.
ALK gene sudden change and/or one " low expression " of gene outcome (labelling as listed in table 1) or " substantially reduce Expression, copy number and/or activity " refer to that the expression of a detection sample or copy number ratio are expressed for assessing or copied The standard error of shellfish number is bigger, at least controls ALK gene sudden change and/or gene outcome (mark as listed in table 1 in sample than one Note) expression, copy number and/or active low twice, three times, four times, five times, ten times or more times, or ratio is several In individual control sample ALK gene sudden change and/or the Average expression level of gene outcome (labelling as listed in table 1), copy number and / or active low twice, three times, four times, five times, ten times or more times.
II. invention implementation
The present invention is at least partly based on the identification to specific region genome, including such as ALK mutant, and in treatment cancer The effect of ALK inhibitor judges.The analysis of ALK gene expressed sequence identifies that the novel mutation to ALK polypeptide is (as listed in table 1 Biomarker, including ALK polypeptide), at least part of polypeptide can be made to have the repellence to ALK inhibitor for treating.Therefore, Different method the most described here exists and/or there is not one or more such biomarker.
In some embodiments, the method for the present invention can be used to monitor the cancer progression of an object, wherein, at cancer progression In may recognize that the sample of object exists one or more sudden change ALK(such as EML4-ALK sudden change), as within the initial some time and In the some time subsequently, the then fewer and feweri response to ALK inhibitor interventional therapy of cancer, vice versa.Implement at other In example, initially between some time and some time subsequently, object has accepted such as chemotherapy, radiotherapy, operation, or other are any useful The treatment such as therapy of suppression cancer, or completed treatment, or alleviated.
Here further illustrate, one or more biomarkers of the present invention (such as sudden change ALK, including sudden change EML4-ALK) Specific identification can be relatively carried out compared with reference sequences, such as SEQ sequence number by genome (such as reproduction and/or the somatic cell) sequence existed 1.Such as, method described here can affect detection biomarker of the present invention, by the DNA to nucleic acid targeting target The amplified reaction that extends include the one or more sudden changes listed by table 1, and analyze one or more mutant nucleic acid targeting mesh of existence Mark realizes.
The most various is it is well known that such as nucleic acid amplification technologies: PCR(polymerase chain reaction), it is disclosed in the U.S. Patent No. 4683195(incorporated by reference), U.S. Patent number 4683202(incorporated by reference) and U.S. Patent number 4800159(draw With reference), and its replacement RT-PCR(RT-polymerase chain reaction), particularly its single step is in patent Disclosed in EP-B-0.569.272, LCR(ligase chain reaction) as open at patent application EP-A-0.201.184, RCR(repaiies Multiple chain reaction) as open (incorporated by reference) at international patent application WO-A-90/01069,3SR(self-sustained sequence replication system) As in international patent application WO-A-90/06995 open (incorporated by reference), NASBA(nucleic acid sequence based amplification technology) as Patent EP-β-0.397.269 and U.S. Patent number 5466586(incorporated by reference) the DNA double chain that uses is template, and TMA (transcript mediated amplification technology) is as at U.S. Patent number 5399491 disclosure (incorporated by reference).
Know the amplification product that can find to exist one or more sudden change in different ways, such as DNA sequencing side in the literature The order-checking of method such as Mulberry lattice and the degree of depth check order, the application of restricted enzyme, allele specific amplification, peptide nucleic acid(PNA) (PNA)-Jie Leading PCR, the discovery of conformational difference, such as chain conformation polymorphic detection (SSCP) and denaturing gradient gel electrophoresis (DGGE), with mark The oligonucleotide probe of note analyzes detection cell membrane (dot blot hybridization), analyzes detection microwell plate, such as reverse hybridization, oligonucleoside Acid linker detection method (OLA, MLPA), first nucleotide change (FNC) technology, crosslinking technological, Rapid Circulation PCR and synchronization Fluorescence analysis (such as 5' nucleotidase/fluorescent quantitation), and with miniature order-checking mass spectroscopy or high performance capillary electrophoresis after PCR.
III. the enforcement that nucleic acid molecules separates
An aspect of of the present present invention relates to isolated nucleic acid molecule, is equivalent to the biomarker of the present invention, including the mark being equivalent to the present invention The polypeptide of note nucleic acid coding or a part of such polypeptide.The nucleic acid molecules of the present invention includes that those nucleic acid molecules belong to be known here Other ALK or ALK-related gene group (such as reproduction and/or somatic cell) and/or coding ALK or ALK-are relevant (such as EML4-ALK) Polypeptide.In certain embodiments, the nucleic acid molecules of the present invention is by including necessary or including core sequence or its fragment listed by table 1 Composition.The isolated nucleic acid molecule of the present invention also includes the nucleic acid molecules being necessarily used for hybridization probe to identify nucleic acid molecules, is equivalent to The labelling of the present invention, including encoding the nucleic acid molecules of the polypeptide being equivalent to labelling, and the fragment of these nucleic acid molecules, as those are fitted Primer or nucleic acid molecules sudden change as PCR amplification.Term used herein above " nucleic acid molecules " be include DNA molecular (as CDNA or genomic DNA) and RNA molecule (such as mRNA) and with nucleotide produce DNA or RNA analog.Nucleic acid molecules Can be strand or double-strand, nucleic acid molecules in certain embodiments be double-stranded DNA.
" separating " nucleic acid molecules is the one separated from other nucleic acid molecules, and it is present in the natural origin of nucleic acid molecules In.In certain embodiments, " separation " nucleic acid molecules is free sequence (such as protein coding sequence), its natural side chain nucleic acid (as sequence is positioned at the nucleic acid of 5' and 3' end) is in the organism genomic DNA that nucleic acid is derived.Such as, in various realities Executing in example, isolated nucleic acid molecule comprises less than 5KB, less than 4KB, less than 3KB, less than 2KB, less than 1KB, less than 0.5KB Or less than the nucleotide sequence of 0.1KB, its natural side chain nucleic acid is in cell-derived and next organism genomic DNA.Additionally, " separate " nucleic acid molecules such as cDNA molecule, substantially there is no other poromerics or culture medium when being produced by recombinant technique, or Substantially precursor or other chemical substances is not had when chemosynthesis.
Statement " does not has other poromerics or culture medium " and includes the nucleic acid molecules of preparation substantially, produces from separation or reorganization Cell microcellular structure is separated.Therefore, nucleic acid molecules substantially do not have poromerics to include that the nucleic acid molecules of preparation has is few In 30%, less than 20%, less than 10%, less than 5%(dry weight) other poromerics or culture medium.
The nucleic acid molecules of the present invention, sudden change ALK gene as listed in table 1, with standard molecular biological technology and number described here Sequence information in recording according to storehouse separates.Using all or part of such nucleotide sequence, the nucleic acid molecules of the present invention can use The hybridization separation of standard and clone technology come isolated (that is, at Sambrook et al., ed., Molecular Cloning: A Laboratory Manual,2nd ed.,Cold Spring Harbor Laboratory Press,Cold Spring Described in Harbor, NY, 1989).
The nucleic acid molecules of the present invention can utilize cDNA, mRNA, or genomic DNA (such as reproduction and/or somatic cell gene group DNA) Expand as the oligonucleotide primers of the PCR amplification technique of masterplate and establishing criteria and obtain.Can be by by DNA sequence analysis method The cloned nucleic acid molecule of amplification is in suitable carrier and express.Additionally, oligonucleotide is equivalent to close fully or partially through standard One-tenth technology is as used the nucleic acid molecules of the automated DNA synthesizer isolated present invention.
In another embodiment, to include having the nucleotide sequence consistent with labelling of the present invention mutual for the isolated nucleic acid molecule of the present invention The nucleic acid molecules of the nucleotide sequence mended or the nucleic acid molecules of the nucleotide sequence of the protein coding consistent with labelling of the present invention.With give The nucleic acid molecules of fixed nucleotide sequence complementary is sufficient and given nucleotide sequence complementary, and it can be with given nucleotide Sequence carries out hybridizing and therefore forms stable double-strand.
Additionally, the nucleic acid molecules of the present invention can only include a nucleotide sequence part, wherein the nucleotide sequence of total length includes this Bright labelling or be equivalent to the coded polypeptide of labelling of the present invention.Such nucleic acid molecules can be used as such as probe or primer.Probe/primer leads to It is commonly used for the oligonucleotide of one or more basic purification.Oligonucleotide generally includes one section of nucleotide hybridized under strict conditions Sequence area, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, extremely Few about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, At least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, extremely Few about 37, at least about 38, at least about 39, at least about 40, at least about 45, at least about 50, at least about 55, at least about 60, At least about 65, at least about 70, at least about 75, at least about 80, at least about 85, at least about 90, at least about 95, at least about 100 or the nucleotide of more continuous print nucleic acid of the present invention.
Probe based on sequence of nucleic acid molecules of the present invention can be used for detecting be equivalent to one or more labelling of the present invention transcribe product or Gene order.Probe includes one group of attached label such as radiosiotope, fluorescent chemicals, enzyme, or enzyme co-factor.This A little probes can act on a part for the diagnosis examination test agent box of the cell or tissue identifying improper expressing protein egg, as right in measured As the level of the nucleic acid molecule encoding protein of cell sample, as detection mRNA level in-site or determine DNA encoding the protein be sudden change or Delete.
The ALK gene of the nucleic acid molecules that present invention additionally encompasses and sudden change or gene outcome (labelling listed by table 1) substantially Cause, such as in they at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85 %, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, At least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5% or higher is identical.In another embodiment, the nucleic acid molecules that present invention additionally encompasses and the ALK base of sudden change Because of or gene outcome (labelling listed by table 1) substantially consistent, such as between them only the most only or at least 1, At least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, At least 12, at least 13, to 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, At least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, At least 31, at least 32, at least 33, at least 34, at least 35, at least 36, at least 37, at least 38, at least 39, extremely Few 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, At least 90, at least 95, at least 100 nucleotide or the difference of any scope.
Term " single nucleotide polymorphism " (SNP) refers to that single core thuja acid occupies polymorphism position, i.e. in allelic sequences There is change in location.The front and back of this position is typically the allelic sequences of high conservative (if sequence variation is less than population 1/100 or 1/1000).SNPs by deleting a nucleic acid on involved allele or can also insert a nucleic acid Cause.Polymorphism position is generally different from and is previously mentioned the substrate of substrate and occupies.Such as, involved allele includes Substrate " T " (thymus pyrimidine) on polymorphism position, " C " (cytosine) that change allele is included on polymorphism position, " G " (guanine), or " A " (adenine).SNP can occur in protein-encoding nucleic acid sequence, in this case, They may cause defect or other muteins, or genetic diseases.So SNP can change the coded sequence therefore of gene Obtain other aminoacid (missense SNP) or SNP can introduce termination codon (meaningless SNP).When SNP does not change albumen The base amino acid sequence of matter, SNP is referred to as " reticent ".SNP may also occur on the noncoding region of nucleotide sequence, and this may result in scarce Fall into protein expression, such as, cause that there is extrastimulation, or may be on the result of protein function impact.
In another embodiment, the isolated nucleic acid molecule of the present invention is at least 7, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60 years old, at least 65 years old, at least 70, At least 75, at least 80, at least 85, at least 90, at least 95, at least 100, at least 125, at least 150, at least 175, At least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 550, at least 650, at least 700 people, at least 800, at least 900, at least 1000, at least 1200, at least 1400, at least 1600, at least 1800, extremely Few 2000, at least 2200, at least 2400, at least 2600, at least 2800, at least 3000, at least 3500, at least 4000, At least 4500, or longer nucleotide, and become to be equivalent to labelling of the present invention or by nucleic acid by making nucleic acid molecular hybridization under strict conditions The coded protein of molecule becomes corresponding labelling of the present invention.Terminology used here " hybridization under strict conditions " refers to for miscellaneous Handing over or the condition of washing, its nucleotide sequence typically at least 60%, 65%, 70%, 75%, 80%, 85% holding is identical each other Hybridization.These strict conditions are at the routine experimentation of the molecular biology of document John Wiley&Sons, N.Y. (1989) 6.3.1-6.3.6 chapters and sections are known and find those technology.Another this, the example of a non-limiting stringent hybridization condition, at 6X At sodium chloride/sodium citrate (SSC) 45 DEG C, subsequently at 0.2X SSC, washed once at 0.1%SDS50-65 DEG C or repeatedly.
Present invention additionally comprises molecular beacon nucleic acid molecule, it at least has the nucleic acid molecule complementary region of and the present invention, this The molecular beacon of sample is used for existing in quantitative sample the nucleic acid molecules of the present invention." molecular beacon " nucleic acid is to include a pair complementary region With there is fluorescence and the nucleic acid molecules of fluorescent quenching related to this.The fluorescence part different from nucleic acid on direction with quencher is relevant, When another complementary region is annealed, the fluorescence of fluorescence is able to quencher by quencher.Another complementary region when nucleic acid molecules When not annealing, the fluorescence quencher lesser extent of fluorescence.Molecular beacon nucleic acid molecule is disclosed, such as, in United States Patent (USP) 5876930(incorporated by reference).
IV. the enforcement of protein and antibody is separated
One aspect, the present invention relates to be equivalent to the separation albumen of separate marking of the present invention and its biological activity.In an embodiment In, ecosystem polypeptide can be by carrying out isolated by the suitable purification schemes of standard protein purification technique from cell or tissue source Be equivalent to the labelling of the present invention.In another embodiment, polypeptide obtains being equivalent to labelling of the present invention by recombinant DNA technology.Weight Group is expressed, and polypeptide is equivalent to use the labelling of the present invention of standard peptide synthesis methods chemosynthesis.
" separate " or " purification " protein or its biologically-active moiety are substantially free of in the cell or tissue source of protein source Cellular material or other contaminating protein matter, or precursor or other chemicals it are substantially free of when chemosynthesis.Statement is " basic Without cellular material " include isolated the cell component of the cell obtained from separation or reorganization prepare protein.Therefore, The protein being substantially free of cellular material includes that the protein of preparation has at least about 30%, at least about 20%, at least about 10%, At least about 5%(dry weight) heterologous protein (" contaminating protein matter " the most mentioned herein).When protein or its biology are lived Property part be that logical restructuring obtains, it can be substantially free of culture medium, such as culture medium at least about 20%, at least about 10%, at least about 5% prepare protein volume.When protein is to be obtained by chemosynthesis, it can be substantially free of the first body of chemistry or other chemistry Thing, the first body of chemistry as involved by from synthetic protein or other chemicals are separated.Therefore, the egg being prepared White matter has ratio additive polypeptide less than about 30%, less than about 20%, less than about 10%, less than about 5%(dry weight) the first body of chemistry Or compound.
Be equivalent to the polypeptide biologically-active moiety of labelling of the present invention and include that the aminoacid sequence by the present invention or pick up from is dashed forward with ALK gene The polypeptide of the protein amino acid sequence composition of change and/or gene prod (labelling listed by table 1).It includes than full length protein more Few aminoacid, and present the vigor at least corresponding to full length protein one times.Under normal circumstances, biologically-active moiety includes one Individual have region or the structure at least corresponding to one times of vigor of protein.The proteins biological activity part of the present invention can be polypeptide, Such as 10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,30,35,40,45, 50,55,60,65,70,75,80,85,90,95,100 or more amino acid lengths.Additionally, delete protein Other active parts in other regions, can be prepared and identify rising of one or more ecosystem polypeptide of the present invention by recombinant technique The active site of effect.
In certain embodiments, polypeptide has the aminoacid sequence of nucleic acid molecule encoding protein listed by table 1.Other useful proteins In matter and these sequences one essentially identical (such as, at least 60, at least 65, at least 70, at least 75, at least 80, At least 85, at least 86, at least 87, at least 88, at least 89, at least 90, at least 91, at least 92, at least 93, at least , and retain different amino 94, at least 95, at least 96, at least 97, at least 98, at least 99, at least 99.5% or bigger) The useful protein active (as ALK inhibitor is had repellence or sensitivity) of full length protein in acid sequence.
Determining the percentage rate characteristic of two aminoacid sequences or nucleic acid, contrast sequence is the most omparison purpose (at first to obtain Aminoacid or protein core acid sequence and second aminoacid or nucleotide sequence junction insert bifurcation point).Then to amino acid residue Or amino acid position corresponding to nucleotide or nucleotide position compare.When first sequence, to be equivalent to second sequence identical Aminoacid residuum or nucleotide take, then the molecule of this position is identical.Percentage rate characteristic in two sequences is that sequence is the most common Enjoy the function (such as % characteristic=same position number #/total positional number #(such as lap position) × 100 of the quantity of position).An enforcement In example, the two sequence is equal length.
Percentage rate characteristic in two sequences can be completed by a kind of mathematical algorithm.It is similar to, non-limiting example, utilizes It can be Karlin andAltschul (1990) Proc.Natl.Acad.Sci.USA that mathematical algorithm compares two sequences 87:2264-2268, improves Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA90:5873-5877 In algorithm.This algorithm refers to the NBLAST of Altschul, et al. (1990) J.Mol.Biol.215:403-410 With in XBLAST method.BLAST nucleotide search can be performed by NBLAST method, score value=100, and word length=12 obtain and this The nucleotide sequence that invention nucleic acid molecules is identical.BLAST protein retrieval can be performed by XBLAST method, score value=50, word Long=3 obtain the amino acid molecular identical with present protein molecule.In order to obtain omparison purpose difference line, may utilize Difference BLAST described in Altschul et al. (1997) Nucleic Acids Res.25:3389-3402.It addition, PSI-Blast can be used as intermolecular remote relationship repeated retrieval occur.When using BLAST, difference BLAST and PSI-Blast During program, the default parameters (such as XBLAST and NBLAST) of each program available (refers to WWW ncbi.nlm.nih.gov On NCBI webpage).In another non-limiting example, the mathematical algorithm for gene comparision is Myers and Miller, (1988) Comput Appl Biosci, the algorithm on 4:11-7.Such a algorithm is incorporated into ALIGN program (2.0 editions) In for comparing amino acid sequence, in PAM120 weight residue table, available difference length points is 12, and difference length points is 4.Separately The one useful algorithm for the identification region of class Sihe linear local sequence is as at Pearson and Lipman (1988) Proc. Fasta algorithm described by Natl.Acad.Sci.USA85:2444-2448.When comparing nucleotide with fasta algorithm Or during aminoacid sequence, such as, PAM120 weight residue table usable levels is the K-unit array of 2.
Percentage rate characteristic between two sequences can determine with or without difference by similar those as described above technology.At meter Calculate in percentage rate characteristic, only have the just calculating of accurately coupling.
The isolated polypeptide or its fragment that are equivalent to the labelling of the present invention can be used as using polyclone and monoclonal standard technique to produce system The immunogen of standby antibody.The antigenic peptide fragment that full-length polypeptide or protein can be used as or the present invention provides is used as immunogen.The present invention Antigenic peptides protein contain at least 8(or at least 10, at least 15, at least 20, at least 30 or more) polypeptide of the present invention The residuum aminoacid of one aminoacid sequence, and include proteantigen epi-position, so that antibody highlights reverse protein structure The immune synthesis clearly with labelling of the present invention is consistent with protein.The enforcement anti-source epi-position that antigenic peptides includes is positioned at protein The region on surface, such as hydrophilic area.Hydrophobicity sequence analysis, hydrophilicity sequence analysis, or similar analysis can be used to identify parent Pool.
Immunogen such as exempts from son, goat, mouse or other mammals typically by immunity inoculation (i.e. immunity) suitably object Or vertebrates prepares antibody.Suitable immune formulation comprises such as recombinant expressed or chemically synthesized polypeptide.Preparation is further also Complete or the incomplete adjuvant including adjuvant such as Freund, or similar immunizing agent.
Therefore, another aspect of the present invention relates to orientation and resists the antibody of polypeptide of the present invention.Term " antibody " and " antibody thing Matter " here can mutually replace, refer to that the immunoactive portions of immunoglobulin molecules and immunoglobulin molecules, i.e. molecule contain Having antigen bond locations, its specificity bonding antigen, such as the polypeptide of the present invention.Special it is adhered on specific polypeptide of the present invention Molecule is as the biological sample containing natural polypeptides is only adhered on polypeptide but is not the most bonded in other molecules in same sample On molecule.The immunoactive portions of immunoglobulin molecules includes F(ab) and F(ab') 2 fragment, its generally applicable employing Enzyme such as pepsin processes antibody and obtains.The present invention provides polyclone and monoclonal antibody.Term used herein " many grams Grand antibody " and " monoclonal antibody composition ", relating to antibody molecule group, it only contains only a kind of has exempting from of specific antigen epi-position The antigen bond locations of epidemic disease ability.
Above-mentioned polyclonal antibody is prepared as in immunogen immune to suitable object with polypeptide of the present invention.In immunization Antibody concentration amount can be by standard technique as measured with enzyme-linked immunosorbent assay (ELISA) in time.If it is required, it is anti- Body molecule can from object (as from the blood of object and serum) acquisition or isolated by known technology such as albumen Matter chromatography purification obtains IgG fragment.Appropriate time the most specific antibody concentration amount after immunity is the highest, antibody-producting cell from Obtain on object and pass through on standard technique such as Kohler and Milstein (1975) Nature256:495-497 public the earliest The hybrid tumor technology opened, human B cell's hybrid tumor technology (see Kozbor et al., 1983, Immunol.Today4:72), EBV-hybrid tumor technology (see Cole et al., pp.77-96In Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, Inc., 1985) or trioma technique prepare monoclonal antibody.For producing hybridoma Technology be well-known (see generally Current Protocols in Immunology, Coligan et al.ed., John Wiley&Sons, New York, 1994).Hybridoma cell produces monoclonal antibody of the present invention by screening hybrid tumor The upper supernatant liquid of culture fluid obtains antibody, and it is bonded on the polypeptide of institute's target, i.e. uses standard ELISA test method.
Preparing the alternative method of monoclonal antibodies-secretion hybrid tumor, the polypeptide of the orientation antagonism present invention can have institute's mesh by screening The restructuring combinatorial immunoglobulin library (as phage antibody displays storehouse) of mark polypeptide identifies and isolated.For producing and screening The test kit in phage antibody display storehouse is commercially available (such as Pharmacia recombinant phages antibody system, production code member 27-9400-01；With Stratagene company SurfZAP phage test kit, production code member 240612).Additionally, it is the suitableeest Share method and the reagent in production with screening antibodies display storehouse can be such as U.S. Patent number 5223409(incorporated by reference), PCT is public Cloth WO92/18619(incorporated by reference), PCT Publication WO91/17271(incorporated by reference), PCT Publication WO92/20791 (incorporated by reference), PCT Publication WO92/15679(incorporated by reference), PCT Publication WO93/01288(incorporated by reference) and, PCT Publication No. WO92/01047(incorporated by reference), PCT Publication WO92/09690(incorporated by reference), PCT Publication WO90/02809 (incorporated by reference), Fox waits (1991) biology/technology 9:1370-1372, Hay et al. (1992) Hum.Antibod. Hybridomas3:81-85, Huse et al. (1989) Science246:1275-1281, Griffiths et al. (1993) EMBO finds in J.12:725-734 waiting.
Additionally, the recombinant antibodies such as chimeric monoclonal antibody with peopleization, including the mankind and nonhuman portions, in the scope of the invention Its available standards recombinant DNA technology interior manufactures.This chimeric and peopleization monoclonal antibody can be by institute in existing document Prepared by the recombinant DNA technology known, as in PCT Publication WO87/02671(incorporated by reference), European Patent Application No. 184187, European Patent Application No. 171496, European Patent Application No. 173494, PCT Publication WO86/01533(incorporated by reference), beautiful State's patent No. 4816567(incorporated by reference), european patent application 125023, Better et al. (1988) Science 240:1041-1043;Liu et al. (1987) Proc.Natl.Acad.Sci.USA84:3439-3443, Liu et Al. (1987) J.Immunol.139:3521-3526, Sun et al. (1987) Proc.Natl.Acad.Sci.USA 84:214-218, Nishimura et al. (1987) Cancer Res.47:999-1005, Wood et al. (1985) Nature 314:446-449, and Shaw et al. (1988) J.Natl.Cancer Inst.80:1553-1559, Morrison (1985) Science229:1202-1207, Oi et al. (1986) Bio/Techniques4:214, United States Patent (USP) 5225539(is drawn With reference), Jones et al. (1986) Nature321:552-525, Verhoeyan et al. (1988) Science 239:1534, and Beidler et al. (1988) J.Immunol.141:4053-4060.
The complete human antibodies being used for treating human subjects needs especially.This antibody can produce with transgenic mouse, and it is incompetent Power expresses endogenous immunoglobulin heavy chain and light chain gene, but can express human heavy chain and light chain gene.Transgenic mouse is just Often selectively antigen immune under mode, as be equivalent to all or part of polypeptide of labelling of the present invention.Orientation antagonism monoclonal anti Body can obtain by traditional hybrid tumor technology.Human immunoglobulin transgenic row again during transgenic mouse B cell is broken up Row existence, and carry out classification conversion and somatic mutation subsequently.Therefore, use this technology, can produce for treating IgG, IgA Antibody with IgE.For producing the technology summary of human antibodies, refer to Lonberg and Huszar (1995) Int.Rev. Immunol.13:65-93.It is discussed in detail and produces this antibody for produce human antibodies and human monoclonal antibody's technology Stipulations, refer to United States Patent (USP) 5625126(incorporated by reference), United States Patent (USP) 5633425 (incorporated by reference), United States Patent (USP) 5569825 (incorporated by reference), United States Patent (USP) 5661016 (incorporated by reference), and United States Patent (USP) 5545806 (incorporated by reference).It addition, company The mankind to non preference antigen are oriented by similar above-mentioned technology as Abgenix, Inc. (Freemont, CA) can be engaged in provide Antibody.
The complete human antibodies of Selective recognition epitope can use the technology being referred to as " guided selection " to produce.This side Method selectivity non-human monoclonal antibodies such as murine antibodies carrys out the complete human antibodies of guided selection identification same antigen epi-position (Jespers et al., 1994, Bio/technology12:899-903).
The antibody (such as monoclonal antibody) of the orientation antagonism polypeptide being equivalent to labelling of the present invention can be used for by standard technique such as affinity chromatograph Or immunoprecipitation carrys out isolated polypeptide.And, this antibody can be used for identifying that labelling is (such as cell lysates or cell suspension Thing) come expression and the model of judge mark.This antibody can also be used for diagnostic monitoring and organizes or internal stream in diagnostic test journey The protein level of body (such as fluid in tumor cell body), as determined the effect of a given therapeutic scheme.Detection can promote to resist Body is coupled on visible material.The most visible material includes but not limited to, various enzymes, prothetic group, fluorescent material, luminescent material, Bioluminescent material and active material.The most suitable enzyme includes but not limited to, horseradish peroxidase, alkali phosphatase ,- Tilactase, or acetylcholinesterase；The most suitable prothetic group compound includes but not limited to streptavidin/biotin, Avidin/biotin；The most suitable fluorescent material includes but not limited to umbelliferone, fluorescein, isosulfocyanic acid fluorescence Element, rhodamine, dichlorotriazine amine fluorescein, dansyl Cl or phycoerythrin；Such as luminescent material includes but not limited to luminol, The most suitable active material includes but not limited to 125I, 131I, 35S or 3H.
V. carrier and the recombinant expressed enforcement of host cell
Another aspect of the present invention relates to carrier such as expression vector, and including the coded polypeptide being equivalent to labelling of the present invention, (or these are many The part of peptide) aminoacid.Terminology used here " carrier " refers to the core having the ability to be transported to its another nucleic acid connected Acid molecule.One type of carrier is " plasmid ", refers to that can increase DNA fragmentation circulation is connected on circular double-stranded DNA.Carrier Another kind of type be viral vector, refer to that increasing DNA fragmentation can connect and be inserted in viral genome.Some carriers are had the ability Independently replicate and introduce in host cell and (such as there is antibacterial and replicate the bacteria carrier and episomal mammalian vectors opened a little).Other Carrier (such as non-add type mammalian vector) is incorporated on host cell gene and introduces host cell, and with host gene one Rise and replicate.Additionally, some carriers, be also expression vector, be have directly express synthesized linker because of ability.In general, The functional expression carrier of recombinant DNA technology may often be such that and obtains from plasmid (carrier).But, plan of the present invention comprises other shapes The expression vector of formula, such as viral vector (such as replication defective retrovirus, adenovirus and adeno-associated virus), they have identical Function.
The recombinant expression carrier of the present invention, including the nucleic acid being suitable for the form expressed in host cell amplifying nucleic acid of the present invention.This meaning Restructuring expressing gene and include one or more control sequence, selectively carry out useful expression based on host cell, and synthesize It is connected on nucleotide sequence express.In recombinant expression carrier, the nucleotide sequence being meant that institute's target of " synthesis connect " with In the control sequence that certain mode is connected to allow nucleotide sequence to express (as transcribed in vivo/translation system in or draw at carrier In host cell when entering host cell).Term " control sequence " refers to control unit containing promoter, enhancer and other expression Part (such as polyadenosine acid signal).This control sequence is such as Goeddel, Methods in Enzymology:Gene Expression Technology vol.185, is described in Academic Press, San Diego, CA (1991).Control Sequence include those nucleotide sequences that direct organization is expressed in polymorphic type host cell and those in some host cells directly The nucleotide sequence (as tissue specificity controls sequence) expressed.By those technical ability in vector expression design document, such as choosing Select host cell to change a little dependence factor, it is desirable to protein expression level etc. be increasingly taken seriously.The expression of the present invention carries Body can introduce host cell and lay eggs next life white matter or peptide, is encoded by nucleic acid described here and has functional protein or peptide.
The recombinant expression carrier of the present invention can be with prokaryotic cell (such as escherichia coli) or eukaryotic cell (as insect cell { has made bar Shape virus expression carrier }, yeast cells or mammalian cell) design the expression of polypeptides being equivalent to labelling of the present invention.It is suitable for Host cell has in Goeddel, supra further to be discussed.Recombinant expression carrier can control such as having T7 promoter Sequence and the in vitro transcription of T7 polymerase and translation.
Procaryotic protein expression is most commonly used that and is instructing pattern of fusion or non-integration albumen containing composition or evoked promoter In the carrier escherichia coli that matter is expressed.Wherein it is usually added into some aminoacid to conduct restructuring in coded protein at pattern of fusion carrier The aminoacid terminator of protein.This pattern of fusion carrier is generally of three effects: 1) increase the expression of recombinant protein；2) Increase the dissolubility of recombinant protein；With 3) by contributing to purifying recombinant proteins at affinity purification active cooperation base. Generally, doing in expression vector merging, proteolytic cleavage sites is at the tie-point introducing fusion part and recombinant protein, It can from the fusion part after pattern of fusion protein purification isolated recombinant protein.This enzyme and their cognate recognition sequence, Including Xa factor, thrombin and enterokinase.Exemplary fusion type expression vector includes pGEX(Pharmacia Biotech Inc;Smith And Johnson, 1988, Gene67:31-40), pMAL (New England Biolabs, Beverly, MA) and fusion The pRIT5 (Pharmacia, Piscataway, NJ) of glutathione-S-transferase (GST), maltose E binding protein, or Protein A, respectively targeting recombiant protein.
Such as, suitable non-fused type coli expression carrier include pTrc (Amann et al., 1988, Gene 69:301-315) and pET11d (Studier et al., p.60-89, In Gene Expression Technology: Methods in Enzymology vol.185,Academic Press,San Diego,CA,1991).The expression of target gene The host RNA polymerase that carrier pTrc relies on from hybrid trp-lac fusion type promoter is transcribed.The expression pET11d of target gene The co expression viral rna polymerase (T7gn1) that carrier relies on from hybridization T7gn10-lac pattern of fusion promoter mediation turns Record.These varial polymerases are positioned at T7gn1 present on prophge by carrying out transcribing of comfortable lacUV5 promoter under control Host's fragment BL21(DE3 of gene) or HMS174 (DE3) offer.
In escherichia coli, one of enforcement maximizing recombinant protein expression is to have Proteolytic enzyme cutting recombinant protein ability Impaired host bacteria in express (Gottesman, p.119-128, In Gene Expression Technology:Methods In Enzymology vol.185, Academic Press, San Diego, CA, 1990).Another enforcement is the core of nucleic acid Acid sequence is inserted in expression vector so that those codons alone in escherichia coli have respective aminoacid (Wada Et al., 1992, Nucleic Acids Res.20:2111-2118).This change of aminoacid sequence of the present invention can be passed through Standard DNA synthesis technique realizes.
In another embodiment, expression vector is Yeast expression carrier.Such as, the carrier for expressing has Saccharomyces Cerevisiae in S bag Include pYepSec1 (Baldari et al., 1987, EMBO J.6:229-234), pMFa (Kurjan and Herskowitz, 1982, Cell30:933-943), pJRY88 (Schultz et al., 1987, Gene54:113-123), pYES2 (Invitrogen Corporation, San Diego, CA), and pPicZ (Invitrogen Corp, San Diego, CA).
It addition, expression vector is rhabdovirus expression vector.It is applicable to protein expression and cultivates insect cell (as Sf9 is thin Born of the same parents) in baculovirus vector include pAc series (Smith et al., 1983, Mol.Cell Biol.3:2156-2165) With pVL series (Lucklow and Summers, 1989, Virology170:31-39).
In another embodiment, the nucleic acid of the present invention is to use the mammalian expression vector in mammalian cell to express. Such as, mammalian expression vector includes pCDM8 (Seed, 1987, Nature329:840) and pMT2PC (Kaufman et al.,1987,EMBO J.6:187-195).When using mammalian cell, the control function of expression vector is usually controlled by virus Element processed provides.Such as, conventional promoter is from polyoma virus, adenovirus 2, cytomegalovirus and simian virus 40.Right Sambrook et al. is referred to, the 16th and 17 chapters and sections in supra in other protokaryon and the suitable expression systems of eukaryotic cell.
In another embodiment, recombinant mammalian expression vector is the ability instructing expression of nucleic acid in specific cell type (as tissue specificity controls element for expression of nucleic acid).Tissue specificity controls element and understands in the literature.Non-limitative example, The tissue-specific promoter being suitable for includes albumin promoter (liver-specific;Pinkert et al.,1987,Genes Dev.1:268-277), lymphoid-specific promoters (Calame and Eaton, 1988, Adv.Immunol.43:235-275), Such as φt cell receptor promoter (Winoto and Baltimore, 1989, EMBO J.8:729-733) and immunoglobulin (Banerji et al., 1983, Cell33:729-740;Queen and Baltimore, 1983, Cell33:741-748), Neuron specific promoter (such as neurofilament promoter, Byrne and Ruddle, 1989, Proc.Natl.Acad.Sci. USA86:5473-5477), pancreas-specific promoter (Edlund et al., 1985, Science230:912-916), With mammary gland specific promoter (such as bovine whey promoter, U.S. Patent number 4873316(incorporated by reference) and European Patent Publication Numbers 264166).Development control promoter also include as mice homeobox protein promoter (Kessel and Gruss, 1990, And-fetoprotein promoter (Camper and Tilghman, 1989, Genes Dev. Science249:374-379) 3:537-546).
Invention still further provides recombinant expression carrier, the DNA being cloned in expression vector including antisense orientation of the present invention divides Son.Being connected to control in sequence it is to say, DNA molecular synthesizes in some way, it allows the antisense of polypeptide of the present invention to compile The RNA molecule of code mRNA expresses (being transcribed by DNA molecular).Synthesis is connected to the control on the nucleic acid molecules of antisense orientation clone Sequence processed can selectively instruct antisense rna molecule continuous expression in the cell of various types of lane, such as, viral promotors and/ Or enhancer, or control sequence can selectively instruct composition, the spy of the typical antisense RNA of tissue-specific or cell The opposite sex is expressed.Antisense expression vector can obtain from recombiant plasmid, phage, or attenuated virus, and its antisensenucleic acids is efficiently Producing under the control of rate control area, its activity can be introduced in carrier by cell type and determine.Use the gene of antisense gene Express control discussion and refer to Weintraub et al., 1986, Trends in Genetics, Vol.1 (1).
Another aspect of the present invention relates to introduce the recombinant expression carrier of the present invention to host cell.Term " host cell " " recombinant host cell " is the most interchangeable.It is reported, these terms are not only related to specific subject cell and also relate to The offspring of these cells or potential offspring.Because some changes can cause generation sudden change or environmental effect subsequently, as actually In the range of offspring is incomplete same with blast cell, but these are still contained in terminology used here.
Host cell can be any prokaryotic cell (such as escherichia coli) or eukaryotic cell (such as, insect cell, yeast or the food in one's mouth Breast zooblast).
Transcribed or rotaring dyeing technology carrier DNA can introduce in protokaryon or eukaryotic cell by traditional.Terminology used here " is transcribed " The original idea " transfected " refers to exogenous nucleic acid be introduced host cell, including calcium phosphate or chlorine by technology known to various documents Changing the co-precipitation of calcium, DEAE-glucosan mediated transfection, fat transfects, or electroporation.For transcribing or being transfected into host cell Appropriate methodology can find on et al. (supra) and other laboratory manuales at Sambrook.
It is known that the stable transfection of right mammalian cell relies on expression vector and technology used, only sub-fraction are thin The genome of born of the same parents can be integrated with foreign DNA.In order to identify and select these intergrants, the selective labelling of gene code is (as right Antibiotic has resistance), it is common that the gene of institute's target is introduced host cell.Typical case implements, and selected marker is introduced those Medicine such as G418, hygromycin and methotrexate medicine are had in the cell of resistance.Have and insert the cytotostatic of nucleic acid transcribe can By selecting medicine to identify (cell of the gene such as with selected marker then survives, and other cells die).
The host cell of the present invention, is equivalent to labelling of the present invention as the protokaryon in culture fluid or eukaryotic host cell can be used to produce Polypeptide.Therefore, the present invention further provides and produce the side of the polypeptide being equivalent to labelling of the present invention with the host cell of the present invention Method.In one embodiment, the method producing labelling includes that the host cell of the present invention cultivating suitable medium (introduces the present invention many The recombinant expression carrier of peptide coding).In another embodiment, method further includes isolated from medium or host cell Labeling polypeptide.
The host cell of the present invention also can be used to produce non-human transgenic animal, such as, in one embodiment, and the present invention's Host cell is the encoded polypeptide sequence introducing in fertilized oocyte or fetal stem cell and being equivalent to labelling of the present invention.Subsequently this Kind host cell can introduce the exogenous array of code book invention labelling in their genome and move for producing Non human transgenic Thing, or change the endogenous gene sequence of the coded polypeptide being equivalent to labelling of the present invention to produce recombinant animal.This animal can be used to Research is equivalent to polypeptide function and/or the activity of labelling, is used for identifying and/or judging polypeptide sliding damper, with the clinic for the treatment of Front test or diagnosis molecule are equally used for finding or identifying labelling, as treated the discovery with diagnostic flag or identification, or pharmaceutical efficacy With specific replacement.
It is non-human animal i.e. mammal used herein of " transgenic animal ", such as rodent, i.e. big rat or mouse, One or more zooblast contains transgenic.Another example, genetic modification animal includes non-human primate, sheep, Canis familiaris L., cattle, sheep, chicken, amphibian animal etc..Transgenic is that foreign DNA is incorporated on the genome of cell, and it comes self-growing The residuum of the genome of transgenic animal and mature animal, therefore in the tissue of one or more cell types or transgenic animal Instruct the expression of encoding gene.It is non-human animal used herein of " homologous recombinant animal ", such as mammal i.e. mouse, It is by changing Homologous integration between the exogenous DNA molecule of endogenous gene and introducing zooblast, i.e. the fetal cell of animal, The early stage of animal is to growing.Transgenic animal are also included within Chan I.T., et al. (2004) J Clin Invest. 113 (4): 528-38and Chin L.et al (1999) Nature400 (6743): those described in 468-72 turn base Because of animal.
The transgenic animal of the present invention can be equivalent to the polypeptide encoding nucleic acid hero to fertilized oocyte of labelling of the present invention by introducing Property protokaryon in produce, i.e. by microinjection, retroviral infection also makes oocyte grow in the jenny of pseudo-fetus. May also comprise the intron sequences of transgenic and polyadenylation signal to improve transgene expression efficiency.Tissue specificity controls sequence The specific cell being connected on transgenic instruct expression of polypeptides of the present invention can be integrated.For producing the method for transgenic animal it is Embryo controls and microinjection, particularly animal such as mouse becomes traditional at document with as at U.S. Patent number 4, and 736,866(draw With reference) and 4,870,009(incorporated by reference), U.S. Patent number 4,873,191(incorporated by reference) and at Hogan, Manipulating the Mouse Embryo,Cold Spring Harbor Laboratory Press,Cold Spring Disclosed in Harbor, N.Y., 1986.Similar method is for the production of other transgenic animal.The animal that transgenic is created can Identify based on transgenic present in the mrna expression of encoded transgene in its genome and/or animal tissue or cell. Then the animal that transgenic is created can be used to breed the animal carrying extra transgenic.Additionally, the animal carrying transgenic can enter one Step breeds other transgenic animal carrying other transgenic.
In order to create homologous recombination animal, the carrier of preparation at least contains the portion gene being equivalent to labeling polypeptide of the present invention coding Delete, increase or replace be introduced into change gene, i.e. upset function.In another embodiment, carrier is designed to this Sample, homologous genes combines, and interior raw gene function upsets (the most no longer encoding function protein, also referred to as " eliminating " carrier).Carry Body may be designed to so, and homologous genes combines, and interior raw gene mutation or other changes but still encoding function albumen are (the most upstream Therefore the change of control area can change interior protedogenous expression).In homology combines carrier, come by increasing the nucleic acid of gene Fill and between the interior raw gene being permitted to be carried in the exogenous gene of carrier and fetal stem cell, occur homology base state in conjunction with carrying out changing section The side chain of 5' and the 3' end of gene.Increase the enough length of side chain nucleotide sequence reach the homology of interior raw gene in conjunction with Purpose.Under normal circumstances, several bases of DNA side chain (all at 5' and 3' end) include in the carrier (referring to Thomas and Capecchi, 1987, Cell51:503for a description of homologous recombination vectors). Carrier is incorporated in fetal stem cell system (as passed through electroporation) and introduces the same of gene and interior raw gene selectable in cell Source is in conjunction with (refer to Li et al., 1992, Cell69:915).During then selecting cell is expelled to the tire bubble of animal (as Mouse) formed polymerization chimera (refer to Bradley, Teratocarcinomas and Embryonic Stem Cells:A Practical Approach, Robertson, Ed., IRL, Oxford, 1987, pp.113-152).Then chimera embryo The pseudopregnant female that tire is injected into being suitable for breeds in animal and embryo grows to childbirth.Homology is nourished again in their embryonic cell Offspring in conjunction with DNA can be used to breeding animals, is transcribed so that containing homology in all of zooblast by the kind of transgenic In conjunction with DNA.For construct homology in conjunction with carrier and homology in conjunction with the method for animal at Bradley (1991) Current Opinion in Bio/Technology2:823-829 and PCT Publication WO90/11354(incorporated by reference), WO91/01140 (incorporated by reference), has more detailed description in WO92/0968 (incorporated by reference), and WO93/04169 (incorporated by reference).
Implementing at another, the animal of transgenic nonhuman can use the selectivity system allowing to control to express of transgenic to produce. One example, such system is the cre/loxP recombinase system of phage P1.Retouching in detail of cre/loxP recombinase system State and refer to Lakso et al. (1992) Proc.Natl.Acad.Sci.USA89:6232-6236.Another example, weight Group system is the FLP recombinase system (O'Gorman et al., 1991, Science251:1351-1355) of saccharomyces cerevisiae. If cre/loxP recombinase system is used to control turning containing CRE recombinase required for transgenes encoding and selectivity protein Gene, the expression of animal.This animal can provide, i.e. by two kinds of transgenic animal by building " double " transgenic animal Hybridization, a transgenic containing encoding selectable protein contains the transgenic encoding recombinase with another.
Non-human transgenic animal described in clone also can be according at Wilmut et al. (1997) Nature385:810-813 With PCT Publication WO97/07668(incorporated by reference) and WO97/07669(incorporated by reference) in disclosed method produce.
V. the enforcement of test kit
Test kit be any products (as packaging or container) include at least one for labelling of the present invention specific detection agents such as Probe, product can be pushed for the unit of the operational approach of the present invention as one, carry or sell.Chemical combination as the present invention Thing, kits and method are used for realizing the ALK mutant gene of present invention during the inventive method and/or gene prod (such as table 1 institute The labelling of row) it is optionally, so that the period to the patient subjects with cancer, the stage, organization type or optimum/ The positive findings acquisition at least about 20%, at least about 40%, at least about 60%, at least about 80% of pre-pernicious/malignant class, at least about 90%, at least about 95%, at least about 99%, or 100%.In certain embodiments, labelling or the marking plate of the present invention are alternative , so that the PPV(positive predictive value of total population) obtain the greater than about the most additional specificity analyses of 10%(more than 99.5%).
The diversification ALK mutant gene of the present invention and/or gene prod (labelling as listed in table 1) are used in the compound of the present invention, Time in test kit and method, the quantity of each labelling, structure and/or activity or expression or copy number can be each many with normal The quantity of unitization labelling, structure and/or activity or expression or copy number compare, in the non-cancer sample of same type, It is single reaction mixture (i.e. with reactant, as each labelling with different fluorescent probes) or is comparable to one Or multiple ALK mutant gene and/or gene prod (labelling as listed in table 1) independent reaction mixture.If diversification ALK Mutant gene and/or gene prod (labelling as listed in table 1) are used, then 2, and 3,4,5,6,7,8,9,10, Or more independent marking can be used or identify.
The present invention includes the chemical combination for sample (i.e. achieving tissue samples or the sample obtained from object) is analyzed cancer cell Thing, test kit and method.These compounds, test kit is substantially the same with the above with method, no matter those, where Needing, this compound, test kit and method are suitable for certain form of sample.
Therefore, the present invention includes having for judgement maybe may have the test kit of the cancer cell that ALK inhibitor reduces sensitivity (i.e. In such as the sample of object samples).Test kit is by one or more ALK mutant genes sufficing to identify out the present invention and/or base Because the reagent of product (i.e. labelling listed by table 1) forms, as be equivalent to ALK mutant gene of the present invention and/or gene prod (i.e. Labelling listed by table 1) nucleic acid or polypeptid specificity bonding include antibody, antibody derivatives, antibody fragment etc..It is suitable for With the nucleic acid that the reagent of nucleic acid (i.e. genomic DNA, mRNA, bonding mRNA, cDNA or similar) bonding includes complementation.Example As, nucleic acid reagent includes the nucleotide (labelling or non-marked) being fixed to substrate, the labeled nucleotide not bonded with substrate, PCR Primer, molecular beacon probe etc..In certain embodiments, test kit contains the useful reagent operating method described here, as At least containing a kind of primer that can identify and hybridize to extend nucleic acid, around at least one nucleic acid extended, at least includes one Mutant listed by table 1 and for detecting the method that there is described mutant amplification targeting nucleic acid.
The test kit of the present invention optionally increases the joint compound useful to the inventive method operation.By such as mode, reagent Box can containing being applicable to complementary nucleic acid annealing or for the liquid (such as SSC buffer) of its specificity conglutinating antibody of protein, one Individual or multiple interval samples, a teaching material describing the inventive method operation, a normal cell sample, a cancer is thin Born of the same parents' sample etc..
The test kit of the present invention can include for detecting labelled protein level or the useful reagent of protein active.Implement at another In example, the test kit of the present invention can include one for detecting the reagent of the methylation of labelling, or includes for detecting labelling knot The reagent of structure change, such as the mutant existed.
VI. prospective medicine
The invention further relates to prospective medicine, including diagnostic detection, pharmacogenomics, clinical trial, they can shift to an earlier date in advance One people of defense sector treatment.Therefore, one aspect of the present invention relates to determining corresponding many with one or more labellings of the present invention Peptide or the quantity of nucleic acid, structure and/or activity, so that it is determined that whether one suffer from cancer or have and suffer from the people of risk of cancer and have Bigger may react ALK inhibitor medicaments therapy.
Therefore, a kind of method that whether a kind of cancer patient of determination of one aspect of the present invention target responds ALK inhibitor therapy. The another aspect of the invention target a kind of method of predictive disease time course.The present invention also has a method to be that target is a kind of pre- Survey the method for contingent critical event in the time course of disease.In certain embodiments, described method includes detecting one The combination of the reactant of individual biomarker or detection biomarker and a kind of ALK inhibitor for treating of the present invention, and detect institute Whether the object stated can produce reaction to described ALK inhibitor for treating.
In certain embodiments, described method relates to the trouble having cancer or suspection to have cancer (as there is cancer symptoms) to diagnosis Gather biological organization sample with person, carry out cytogenetics examination, detection ALK sudden change (as listed in table 1 those).
Described screening method and the result of explanation thereof can predict that patient is to ALK inhibitor (such as PF-02341066 and/or PDD) The reaction for the treatment of.As described herein, the existence of a kind of ALK sudden change shows not produce, with those, patient's phase that a kind of ALK suddenlys change Ratio, ALK inhibitor (such as PF-02341066 and/or PDD) therapy can more effectively treat cancer cell.
In one embodiment, method of the present invention includes contacting with a DNA sample, such as a germ cell line or body Cell genomic dna, such as a chromosome sample, it is that the cell separation of collection is polynucleotide probes from patient, these Probe can be used for specifically under strict conditions with genomic DNA hybridization, make the chromosome sequence generation cell in patient's cell lose Pass and learn abnormal (ALK as described in the present invention sudden change).These results analyzed can predict patient for treatment's reagent, especially ALK Inhibitor (such as PF-02341066 and/or PDD) treats issuable reaction.
In another embodiment, there is the time interval of critical event during determining patient disease, thus calculate a time Process, wherein said calculating can predict whether a patient has a long time process.In another first draft example, Described critical event is to develop into death from ID.In another embodiment, described critical event is to examine from for the first time The disconnected disease that develops into is transplanted.In another is implemented, described critical event is to develop into deteriorate from ID.At another In embodiment, described critical event is to transplant from disease to develop into death.In another embodiment, described critical event It is to transplant from disease to develop into deteriorate.In another embodiment, described critical event is to develop into death from deterioration.One In a little embodiments, described time course is to use whole survival rate, development time and/or use RECIST or other reaction normal Measure.
In certain embodiments, the independent clinical samples at least two patient population can form a prediction measurement.Implement at some In example, group's number is two, and therefore clinical samples is divided into a patient population having an ALK sudden change and a neither one ALK The patient population of sudden change.In certain embodiments, the ALK mutation content in object both with have what ALK suddenlyd change to compare, again and do not have Comparing of ALK sudden change；If described patient has a kind of ALK sudden change, then patient is unlikely to a kind of ALK inhibitor (such as PF-02341066 and/or PDD) produces reaction and/or patient unlikely has a long time process.At some In embodiment, group's number is more than two, includes three groups, four groups, five groups and six groups without limitation, according to prediction ALK The layering of inhibitor effect and the relation of special ALK sudden change.In some embodiments, it may be possible to reaction be to use whole survival rate, Development time and/or use RECIST standard are measured.In certain embodiments, ALK inhibitor be PF-02341066 and/ Or PDD.
Another aspect of the present invention target is a kind of determines that an object suffering from ALK sudden change positive cancer is the most likely to one Plant the method that the time course of ALK inhibitor (such as PF-02341066 and/or PDD) generation reaction and/or disease is the longest. A kind of method of another aspect of the invention target, it may be used for predicting weight in the object suffering from ALK sudden change positive cancer Want the probability of event.
1. the method for detection ALK sudden change
Those of ordinary skill in the art is generally known to detect ALK gene sudden change and/or gene outcome (labelling as listed in table 1) Method includes hybridizing substrate detection.Such as, in one sample of detection, the method for the copy number of coding nucleotide includes one Southern blotting.In a kind of Southern blotting, described genomic DNA (generally spreads and separates at a kind of electrophoresis On gel) and the probe hybridization of a specific target sequence.The strong hybridization signals that the probe of comparison object sequence sends is with normal MRNA(is the most identical or relevant cell, tissue, a non-reinforcing part in organ etc.) analyze the control probe signals energy sent Detect whether target polynucleotide exists, and detect its copy number.Or a kind of Northern blotting can be used to detect Coding nucleotide copy number in one sample.In a kind of Northern blotting, mRNA and the probe of specific target sequence Hybridization.The strong hybridization signals that the probe of comparison object sequence sends is the most identical or relevant cell with normal mRNA(, tissue, device A non-reinforcing part in official rank) analyze the control probe signals that sends and can detect whether target polynucleotide exists, and detect Go out its copy number.
Determine a kind of selective method of copy number be in situ hybridization (such as Angerer (1987) Meth.Enzymol152: 649).In situ hybridization generally includes following step: (1) fixing organization or biological structure are used for analyzing；(2) pre-treat biological structure To improve the accessibility of target dna, and reduce non-specific binding；(3) by mixture of ribonucleotides and described biological structure or group Nucleotide hybridization in knitting；(4) post-hybridization washing falls those nucleotide fragments being not integrated in hybridization；(5) detection hybridization core Acid fragments.The reagent used in each step above-mentioned and condition can be changed according to reality application.
Preferably the detection of hybridization substrate is non-limiting includes existing " direct probe " method, such as Southern blotting or the most miscellaneous Friendship method (as FISH and FISH adds SKY), and " comparing probe " method, such as comparative genome hybridization (CGH), such as cDNA Substrate or oligonucleotide substrate CGH.Described method can use in every way, and it includes that substrate is (as thin without limitation Film or glass) combined techniques or matrix substrate method.
On the one hand it is to use fish analysis.Cell sample can be according to method known to a person of ordinary skill in the art with patient Obtain, detect by one known to a person of ordinary skill in the art suitable Hemapoiesis detection method, such as FISH method.One In individual embodiment, FISH can be according to VysisTMSystem (Abbott molecule) operates, and the operation instruction of its producer can become Reference for the present invention.
Probe for using contains DNA fragmentation, its with chromosome dyad present in DNA substrate sequence complementary.Bittner etc. Two parts of United States Patent (USP)s of the Vysis company of invention, its patent No. 5,491,224(incorporated by reference) and 6,277,569(quote ginseng Examine) disclose the probe example that the present invention uses, and these probe labellings are hybridized in sample.
The length of chromosome probe ordinarily be about 50-105.Longer probe generally includes the more small pieces of a length of 100-500 nucleotide Section.It is commercially available with the probe of centromeric DNA and locus specificity DNA hybridization, such as Vysis company (Tang Nasi Ge Wei, Illinois), molecular phycobiliprotein complexes (Liu Zhen, Oregon) or Cytocell (Oxfordshire, Britain).Visit Pin can also optionally use chromosome set or genomic DNA to be made by standard technique rather than commercially prepared.Such as, The DNA source that can use includes genomic DNA, and the DNA sequence of clone, the body containing the part in or is thin Born of the same parents hybridize, the normal chromosomal complementation of chromosome (such as human body chromosome) and host and flow cytometer or the dyeing of micro-purification Body.Described insertion sequence can pass through clone and separate, or expands separation with the site-specific of polymer chain reaction (PCR). See, for example, Nath and Johnson, Biotechnic Histochem., 1998,73 (1): 6-22, Wheeless et Al., Cytometry1994,17:319-326 and United States Patent (USP) 5,491,224(incorporated by reference).
Can determine that whether this sequence exists cytogenetic abnormalities with the probe of a Genomic signature hybridization.Cytogenetics Learning one of exception is a deletion.Although these deletions can be one or more complete chromosome, but they are usually directed to one Individual or the excalation of multiple chromosome.If the whole sequence of a chromosome contained in probe is deleted from a cell, So this probe generally will not again with the DNA hybridization in cell, then would not produce signal on chromosome.If an office Portion contains the chromosome sequence of a probe and deletes from a cell, then this probe still can be with the DNA in this cell Hybridization, but signal can reduce.A such as signal loss than with control cell DNA carry out probe hybridization do not contain spy The gene unconventionality of detection estimated by pin.In certain embodiments, cytogenetic abnormalities can produce at least 1,5,10,20,30,40, 50,60,70,80,90,100,110,120,130,140,150,160,170,180,190,2 00 or more Cell.
The cytogenetic abnormalities that can detect includes non-reciprocal translocation, interior chromosome inversion, point mutation without limitation, lacks, Copy number changes, gene expression dose change and germline mutants.A kind of particularly cytogenetic abnormalities is a kind of duplication. These duplications can be whole chromosome set, it is also possible to is the sequence less than a complete chromosome.If one contains one The chromosome sequence of probe is replicated in a cell, then probe and this cell DNA hybridize and there is not the dye containing probe The signal number controlling to produce in cell of colour solid sequence is compared, it will usually at least produce an extra signal.Although detection human body Any probe of chromosome set 2p23 or its ortholog or any chromosome sequence contains the ALK of a 2p23 or its ortholog Group translocation, but it can also use.Prior art has been disclosed for suitable probe (as (Tang of Vysis company can be used Na Sigewei, Illinois) produce).
Chromosome probe can be with labelling, so the hybridization of chromosome sequence can detect.Probe generally can be with one Fluorogen, a direct labelling of organic molecule, wherein organic molecule can be sent out glorious after absorbing low ripple/high energy light.Described fluorescence Group makes probe need not second detection molecules just can become visible.After one fluorogen and a nucleotide covalent bond, Described nucleotide can directly use standard technique, and such as lack transcription, the realization such as random primer and PCR labelling is combined with probe. Or Deoxydization nucleotide and probe can be made to promote to turn ammonia with a connection.Described fluorogen can be with the deoxynucleoside promoted after turning ammonia Acid covalent bond.See United States Patent (USP) 5,491,224(incorporated by reference).
United States Patent (USP) 5,491,224 discloses and is labeled as the probe of some cytosine residues and has a fluorogen labelling covalent bond. The quantity of fluorogen labelling cytosine substrate enough just can produce a detectable fluorescence signal when one, therefore labelling DNA fragmentation substantially can retain special complementary key (hybridize) characteristic relevant to the chromosome set that can detect or chromosome sequence. These probes can be prepared: attracts unlabelled DNA probe sequence, with a linking group promote in the sequence to turn ammonia some Deoxydization nucleotide, by a fluorescent labeling and at least some of deoxycytidine substrate covalent bond promoting to turn ammonia.
Probe can also use lack transcription, random primer or PCR labelling to carry out labelling.Labelling both can use fluorogen (directly) Labeled nucleotide, it is possible to use hapten (indirectly) labeled nucleotide completes.The non-limiting representative instance of labelling includes: AMCA-6-dUTP, cascade indigo plant-4-dUTP, glorious element-12-dUTP, rhodamine-6-dUTP, texas Red-6-dUTP, Cy3-6-dUTP, Cy5-dUTP, biotin (BIO)-11-dUTP, digoxin (DIG)-11-dUTP or dinitro benzene (DNP)-11-dUTP。
Probe can also be with biotin or digoxin labelling indirectly, it is also possible to radiosiotope, such as labellings such as 32p and 3H, Although then needing for the second detection molecules or further process identifying probe.Such as, the probe labelling of a biotin Avidin and a detection labelling can be utilized to combine and detect.Such as, avidin can be combined with an enzyme labelling, Such as alkali phosphatase or horseradish peroxidase.Enzyme labelling can use a substrate of enzyme and/or catalyst to react at standard colorimetric Middle detection.The catalyst of alkali phosphatase includes 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium.Diaminobenzene Formic acid can use as the catalyst of horseradish peroxidase.
Before probe can also be prepared as making hybridization or hybridization in, a fluorogen or other labelling will not be a part of DNA, and Increasing detection this probe hybridization after hybridization is a chromosome set.Such as, the probe of use can have be combined with DNA anti- Original molecule.After hybridization, these antigen molecules can use specific antibody to react with antigen molecule and detect.These antibody self can To be combined with a fluorogen, or a second antibody detection with a covalency fluorogen can be used.
But after Chu Liing or after modification, described DNA probe can carry out common purification before hybridization uses, the most anti-to remove The resultant product (fluorophore molecule as not having and DNA combines) answered.
Method known to a person of ordinary skill in the art can be used before hybridization to make chromosome probe degeneration.Hybridization step generally wraps Include: in label probe mixture, add the DNA that blockades of a kind of excess, probe will be blockaded and be used for detection under hybridization conditions Chromosome sequence contacts, as in a solid phase, and DNA therein degeneration, rinse non-hybridized probe, detect and dye The probe that body or chromosome sequence combine.
Probe hybridizes under hybridization conditions or is annealed into described chromosomal DNA." hybridization conditions " be advantageous for a probe with The condition of annealing between target chromosome DNA.Although the annealing of different probe is according to probe length, the difference of the situations such as concentration of substrate And different, prior art has been disclosed for the various concentration and probe concentration beneficially annealed, hybridization temperature, salinity and other factors.
Various concentration, substrate mixture, complex and probe length, and salinity, temperature and temperature retention time can promote miscellaneous Hand over.Such as, in situ hybridization is generally carried out in hybridization buffer, and described hybridization buffer contains 1-2x SSC, 50-65% Methanamide and the DNA that blockades preventing Non-specific hybridization.Above-mentioned hybridization conditions generally includes the temperature of about 25 DEG C to 55 DEG C, about The temperature retention time of 0.5 to 96 hours.
Various rinsing can be used to remove the non-specific binding formed between chromosome probe and the outer DNA of target sequence.Change Temperature and the salinity of rinsing strictly controls these rinsings every time.Such as under conditions of very strict, the temperature of rinsing is about 65-80 DEG C, use the SSC of 0.2x to about 2x, and the nonionic detergent of about 0.1%-1%, such as Nonidet P-40
(NP40)。
Stringent condition when rinse temperature when increasing rinsing or salinity can reduce rinsing.Need weight of blockading in some applications The amount of hybridization of complex sequences.TRNA, human genome DNA or Cot-I DNA can be used the most in certain embodiments to blockade Non-specific hybridization.
After rinsing, described microscope slide can be finished and air is dried, and is subsequently adding medium, use a kind of dyeing liquor such as DAPI with One coverslip.Observe microscope slide immediately and before check, be stored in-20 DEG C.
In order to use fluorescent probe in fluorescence in situ hybridization (FISH) technology, can be that each fluorescence arranges a band fluorescence mistake The fluorescence microscopy mirror device of filter, thus it can be seen that fluorescence, or uses double or three passband filter plants to observe many fluorescence. See, for example, United States Patent (USP) 5,776,688(incorporated by reference).Or the technology such as flow cytometry such as can also be used Check the Crossing system of chromosome probe.FISH can be used to detect the sequence of resurveying of chromosomal copy number or chromosome.These The hybridization of probe and complementary DNA or combination, owing to they marked fluorescence labels, therefore researcher can use a fluorescence to show Micro mirror observes the position of these DNA sequence.Different from other technology of great majority used in chromosome research, FISH need not Cell is that activity separates, and it is to carry out in the cell of non-subregion, is therefore the method for a high-efficiency multi-function.Therefore FISH Subregion cell can be used to realize, or use subregion cell or Cellular compartment circulation to realize.U.S. of Gray and Pinkel State's patent 5,447,841 discloses the many technology in fish analysis.
FISH result be referred to control cell illustrate design probe in detecting, wherein control cell be known to do not contain special Cytogenetic abnormalities.By described probe with control DNA in cell and carry out the method for FISH hybridization and probe of the same race and do not test Or the DNA hybridization in the cell of assessment specific cell genetic alteration compares.When the probe of a design is for detection one During the disappearance of individual chromosome or chromosome sequence, the most described probe with do not test or assess specific cell genetic alteration DNA hybridization in cell ratio is less with the DNA hybridization controlled in cell.Usually not one probe letter in these test cell Number, the chromosome sequence disappearance that probe hybridizes the most therewith is described.When the probe in detecting of a design is to a kind of dyeing body weight Multiple or increase, then the most described probe and the DNA hybridization in cell the most after tested than with the DNA hybridization controlled in cell More.Generally having an extra probe signals in test cell, an extra chromosome sequence is described, it would generally Hybridize with probe.
In CGH method, first accumulation regions of nucleotide (as from a sample, such as a possible tissue) indicates one Individual first labelling, when second accumulation regions (such as a control, the cell/tissue as healthy from) of nucleotide indicates one Individual second labelling.The hybrid rate of these nucleotide is by the combination rate of in matrix two kinds of (first and second) labellings Yu each optical fiber Determine.At chromosome deficiency or duplication, signaling rate and two kinds of mark of detection are different, and this can detect copy number.Matrix substrate CGH can also use single color mark realize (use two kinds of dyestuffs to come differently marking of control and possible tissue samples, And before hybridization, mix them, owing to competitive hybridization can produce a ratio on matrix).In single color CGH, Can to control labelling hybridization be a readable matrix and complete signal by described, it would be possible to the hybridization of tissue samples labelling for can First matrix (having identification content) read and complete signal.Complete signal in two matrixes calculates different copy numbers.
As at Albertson (1984) EMBO J.3:1227-1234;Pinkel(1988)Proc.Natl.Acad.Sci. USA85:9138-9142；European Publication Number 430,402；Methods in Molecular Biology,Vol.33:In situ Hybridization Protocols, Choo, ed., Humana Press, Totowa, N.J. (1994) etc. have been disclosed for It is adapted in use to the crossing scheme of the method for the invention.In one embodiment, Pinkel, et al. (1998) Nature is employed Genetics20:207-211 or Kallioniemi (1992) Proc.Natl Acad Sci USA89:5321-5325 (1992) Disclosed hybrid method.United States Patent (USP) 6,455, discloses matrix backplanes CGH in 258, the most all of content can be as the present invention Reference.
In another embodiment, it is possible to use presence or absence and copy number are measured in amplification substrate assessment.Substrate is amplified at this In assessment, described nucleotide sequence is as a template in amplified reaction (such as polymerase chain reaction (PCR)).One In individual quantitative amplification, the amount of amplified production is proportional to the template amount in original sample.Proper control, such as health tissues Described copy number can be measured.
All known " quantitatively " TRAP of those of ordinary skill in the art.Such as, quantitative PCR is directed to use with identical primer together Time one known quality of coamplification control sequence.Which provides an internal standard that can be used to calibrate PCR reaction. Innis,et al.(1990)PCR Protocols,A Guide to Methods and Applications,Academic Press, Inc.N.Y. have been disclosed for the concrete scheme of quantitative PCR.Ginzonger,et al.(2000)Cancer Research60:5405-5409 has been disclosed for the copy number using quantitative PCR analysis to measure DNA in microsatellite.Gene Nucleotide sequence enough make a those of ordinary skill in the art conventionally to select amplification gene is any portion of to be drawn Thing.Quantitative fluorescent PCR can also be used in method of the present invention.In quantitative fluorescent PCR, it is quantitatively to believe based on fluorescence Number quantity, such as TaqMan and sybr gene.
Other suitable TRAP includes that ligase chain reaction (LCR) (sees Wu and Wallace (1989) without limitation Genomics4:560,Landegren,et al.(1988)Science241:1077,and Barringer et al.(1990) Gene89:117), transcription amplification (Kwoh, et al. (1989) Proc.Natl.Acad.Sci.USA86:1173), Automatic continuous gene replication (Guatelli, et al. (1990) Proc.Nat.Acad.Sci.USA87:1874), Point PCR and the adaptive thing PCR of connection etc..
Loss of heterozygosity (LOH) figure (Wang, Z.C., et al. (2004) Cancer Res64 (1): 64-71;Seymour,A. B.,et al.(1994)Cancer Res54,2761-4;Hahn,S.A.,et al.(1995)Cancer Res55, 4670-5;Kimura, M., et al. (1996) Genes Chromosomes Cancer17,88-93) can be used for knowing Other extension increasing sequence or deletion sequence.
2. the method assessing gene expression
The level of marker expression can also be assessed.The expression of a labelling of the present invention can also be with various known detections one The individual method transcribing molecule or albumen is assessed.The limiting examples of these methods includes for detecting secretion, cell surface, Cytoplasm or the immunological method of nucleoprotein, protein separation method, protein function or Activity determination, nucleic acid hybridization, Nucleic acid reverse transcription method and nucleic acid amplification.
In certain embodiments, the activity of a specific gene can turn by measuring by measuring genetic transcription (such as mRNA) Record the quality of albumen or characterize by measuring gene product activity.Marker expression can monitor by various methods, including passing through Detection mRNA level in-site, protein level or protein active etc. can use standard technique to measure.The content of detection includes base Because of expression amount (such as genomic DNA, cDNA, mRNA, protein or enzyme are lived) or can be the one of gene expression dose Individual evaluation quantity, especially compared with a control level.The type of this detected level can be released from the context.
Those of ordinary skill in the art (see supra such as Sambrook) all known those use hybridization techniques to detect and / or the method for quantitative to genetic transcription (prepared by mRNA or cDNA here).Whether such as a kind of assessment cDNA exists and measures Method include Southern blotting of the present invention.In brief, described mRNA is separated (as used a kind of acid Guanidine-phenol chloroform, the supra such as Sambrook) and reverse transcription be product cDNA.Then described cDNA optionally absorbs And in buffer, run gel, it is transplanted to thin film.Then the nucleotide probe special with target cDNA is used can to complete hybridization.
One ultimate principle of these diagnosis and forecast assessment includes preparing a kind of sample or reactant mixture, and they can be containing one Individual labelling and probe interact and combine, thus form a complex, and it can move from reactant mixture and/or delete. These assessments can be implemented by various methods.
Such as, it is labelling or probe to be fixed in a solid phase support that one of which realizes the method for assessment, or as one Substrate, and target label/probe that detection is fixed in solid phase at the end of reaction.In an embodiment of this method, from Collecting sample on object, assesses its concentration whether having labelling and labelling, and is fixed in a carrier or solid phase support. In another embodiment, can be contrary situation, its middle probe be fixed in a solid phase, and one gathered on object Sample can be as a non-frozen composition reaction in assessment.
Prior art discloses many methods be fixed in a solid phase by assessment composition.They include without limitation will be in conjunction with Biotin and the fixing labelling of streptavidin or probe.This biotinylated assessment composition can utilize prior art Known in, technology is (such as biotinylation kit, biotinylation kit, Pierre's Si chemical industry, Rockford, Illinois State) prepared by biotin-NHS (N-hydroxy-succinimide), and fix at the hole of streptavidin coating 96 orifice plate (Pierre's Si chemical industry).In certain embodiments, these surfaces securing assessment composition can be prepared in advance and preserve.
Other suitable carrier or the solid phase support that use in these assessments include any to be combined with level molecule belonging to labelling or probe Material.Known support or carrier include glass, polystyrene, nylon, polypropylene, polyethylene without limitation, and Portugal gathers Sugar, amylase, natural and modified cellulose, polyacrylamide, gabbro and magnetic iron ore.
In order to make to realize assessment in aforementioned manners, loose composition is added in the solid phase having secured the second composition.Instead After should completing, not compound composition is removed (such as rinsing) under conditions, makes any composite parts be retained in regularly in solid phase. Certain methods of the present invention can be used to be deleted by the labelling/probe complex being fixed in described solid phase.
In another embodiment, when described probe is uncured assessment composition, it can indicate of the present invention Detectable label known to the those of ordinary skill of field, such that it is able to be used directly or indirectly in realization assessment and read assessment result.
Can also directly detect labelling/probe complex composition, without operation or two kinds of compositions of labelling (labelling or probe) again, Such as utilize fluorescence energy transfer techniques (for example, see United States Patent (USP) 5,631,169(incorporated by reference of Lakowicz etc.)).Choosing Selecting a fluorescent labeling on first " donation " molecule, then use the light of suitable wavelength to excite, the fluorescence launched can be by one A fluorescent labeling on individual second " acceptance " molecule absorbs, due to described absorption energy so can fluoresce successively.Or Described " donation " protein molecular can be simply by the natural fluoresence energy of trp residue.Selected labelling can send not The light of co-wavelength, makes " acceptance " molecular marker can be different from " donation ".Since the efficiency of transfer is divided with molecule between labelling From distance dependent, then intermolecular space interval can also be evaluated.When intermolecular generation combines, assessment " accepts " The time of the phosphor persistence that molecule sends is the longest.One FET combines situation and even can enter by known standard fluorescence detection method Row conventional sense (as used a kind of fluorescent agent).
In another embodiment, determine that the ability of one labelling of a probe identification can need not labelling or assessment composition (spy Pin or labelling) realize, but utilize a kind of technology, as real-time biomolecular interaction analysis (BIA) (see as Sjolander,S.and Urbaniczky,C.,1991,Anal.Chem.63:2338-2345and Szabo et al., 1995,Curr.Opin.Struct.Biol.5:699-705).As described herein, " BIA " or " surface plasma Resonance " be a kind of in situ study biologic specificity interact technology, and do not have any interaction of labelling (as BIA clone). Knots modification on mating surface (interaction of an integrated structure) can make the optical index (surface plasma of near surface The optical phenomena (SPR) of resonance) change, produce a detectable signal, the reality that it can react as a kind of biomolecule Time indicator use.
Or in another embodiment, similar diagnosis and assessment in advance can use labelling and probe in a kind of liquid phase As solvent.In such a is assessed, differential centrifugation can be included without limitation by more any standard techniques, chromatography, Electrophoresis and immunoprecipitation separate described compound token and probe.In differential centrifugation, labelling/probe complex can use one The assessment composition that Series Centrifugal step is never combined is separated, because the different size of complex and density make the heavy of them Fall balance also differ (for example, see Heegaard, N.H., 1998, J.Mol.Recognit.Winter11 (1-6): 141-8; Hage,D.S.,and Tweed,S.A.J Chromatogr B Biomed Sci Appl1997Oct
10;699 (1-2): 499-525).Standard chromatographic techniques can be used for never isolating compound molecule in complex.Such as, solidifying Glue filtration chromatography separates molecule according to molecular size, and it uses a kind of suitably gel filtration resin in a post, the most relatively Big complex can be separated from less non-composite parts.Similarly, compared with non-composite parts, there is different electricity Labelling/the probe complex of lotus performance can be used as being different from the complex of non-composite parts, such as by using ion to exchange Laminate resin.Those of ordinary skill in the art's these resins known and chromatographic technique (for example, see Heegaard, N.H., 1998, J.Mol.Recognit.Winter11(1-6):141-8;Hage, D.S., and Tweed, S.A.J Chromatogr B Biomed Sci Appl1997Oct10;699(1-2):499-525).Composite valuations composition can also never be tied by gel electrophoresis The composition closed is separated (for example, see Ausubel et al., ed., Current Protocols in Molecular Biology,John Wiley&Sons,New York,1987-1999).It is such as according to albumen and core in this technology The size of thuja acid complex and electric charge separate theirs.In order to make electrophoresis process keeps lasting combination, do not reduce The undenatured gel matrix material of reagent and condition are typical.The suitable bar of specific assessment known in those of ordinary skill in the art Part and composition.
In a preferred embodiment, mRNA be equivalent to the level of labelling can use method well known in the art in position or Test tube determines.Term " biological specimen " can include tissue, cell, biofluid and separator thereof, and they are right from one As gathering acquisition with it, and organizing present on an object body, cell is the same with liquid.In test tube method, any RNA divides Will not select to prevent mRNA from separating from technology, it can be used for from cell purifying RNA (for example, see Ausubel et al., ed., Current Protocols in Molecular Biology, John Wiley&Sons, New York1987-1999). Furthermore, it is possible to prepare substantial amounts of tissue samples, and use method known to a person of ordinary skill in the art to process them, such as that Get Ya Lei mono-step RNA partition method (1989, United States Patent (USP) 4,843,155 (incorporated by reference)).
The separating nucleotide that can use in hybridization or amplification assessment includes that Southern or Northern analyzes without limitation, Polymer chain type response analysis and probe matrix.A kind of diagnostic method detecting mRNA level in-site include by separate mRNA and one Nucleic acid molecule (probe) separates, and then the mRNA with detection gene code hybridizes.Described nucleotide probe can be such as A kind of global cDNA or one part, as length is at least a kind of few of 7,15,30,50,100,250 or 500 nucleotide Nucleotide, it can specific hybridization be enough a mRNA encoding a labelling of the present invention or genome under strict conditions DNA.Other suitable probe can also use in diagnostic assessment of the present invention.The hybridization of the mRNA of one band probe is said Labelling in bright problem is expressed.
In an example, mRNA is fixed on a surface of solids and contacts with a probe, such as at an agarose gel The upper mRNA running separation, and mRNA is transferred to thin film from gel, such as nitrocellulose.In another example, by probe It is fixed on a surface of solids, mRNA is contacted with probe, such as at the Genechip array of an Affymetrix.One Known mRNA method can be used for detecting mRNA level in-site by individual those of ordinary skill, and wherein said mRNA is by institute of the present invention State label coding.
Described probe can be full length nucleotide sequence or the nucleotide sequence coded albumen less than total length.Shorter probe can To detect specificity by rule of thumb.Such as preferably nucleotide probe has 20 bases or length longer (for example, see Sambrook et al.for methods of selecting nucleic acid probe sequences for use in nucleic acid Hybridization).Hybrid protein can detect whether cDNA quantitatively.
A kind of process for selective determining transcriptional level corresponding with a labelling of the present invention in sample includes amplification oligonucleotide Method, as utilized rtPCR(Mullis, the United States Patent (USP) 4 of 1987, the experimental example (incorporated by reference) listed by 683,202), even Connect enzyme chain reaction (Barany, 1991, Proc.Natl.Acad.Sci.USA, 88:189-193), automatic continuous sequence Replicate (Guatelli et al., 1990, Proc.Natl.Acad.Sci.USA87:1874-1878), transcription amplification system System (Kwoh et al., 1989, Proc.Natl.Acad.Sci.USA86:1173-1177), Q-β replicative enzyme (Lizardi Et al., 1988, Bio/Technology6:1197) roll recursive copying (Lizardi etc., United States Patent (USP) 5,854,033 (incorporated by reference)) or other any amplification oligonucleotide method, subsequently by using technology known to a person of ordinary skill in the art Detection amplifier molecule.Fluorescence rtPCR can also use in method of the present invention.In glorious rtPCR, it it is quantitatively root According to the amount of fluorescence signal, such as fluorescent quantitation and fluorescent dye.These detection schemes are particularly useful for the core that amount detection is the least Thuja acid molecule.As described herein, amplimer is confirmed as a pair nucleic acid molecule, and they can be annealed to the 5 ' or 3 ' of gene End (increase respectively and reduce chain, or vice versa as the same), and between contain a short sequence.Amplimer normal length is 10-30 Individual nucleotide, its both sides have one to be about 50-200 nucleotide sequence.In amplification condition and amplifing reagent, these primers make one The amplification of individual nucleic acid molecule includes the nucleotide sequence of primer both sides.
In method, mRNA need not separate from cell before detection in position.In these methods, it is possible to use known miscellaneous A cell or tissue sample is prepared/obtained to friendship method.Then described sample is fixed on one support, usually one Glassy solids, then contacts with a probe, and this probe can be with the mRNA hybridization encoding described labelling.
Owing to needing optionally to predict according to the limiting expression level of described labelling, therefore can also be according to described labelling Specification expression determine.Expression can generally be revised the specification expression of a labelling and carry out specification, is expressed Level, compared with cold gene expression dose, can be expressed such as a house-keeping gene etc..The Suitable genes of specification includes House-keeping gene, such as actin gene or epithelial cell specific gene.This specification makes a sample, as one to decent This can be compared with another sample, the such as expression in a non-cancer stricken sample, or can compare separate sources Expression between sample.
Or expression can also be as a relative expression.In order to determine the relative expression levels of a labelling, Can compare with the cancer cell sample separated with 10 or more specification sample before determining the expression of problem sample Relatively obtain the expression of described labelling, or putting down even with each gene assessed in 50 or more multisample great amount of samples All expressions, and as a basal level expression of described labelling.May determine that detection sample (limiting expression level) The described marker expression level then labeled mean expression value obtained distinguished.This generates a relative expression levels.
In certain embodiments, basis determines that the sample of middle use can take from cancer cell or the specification cell of homologue type. Cell derived can select according to the relative expression levels used.The expression found in operating specification tissue is average as one Express composition acid be confirm assessment mark whether be specific to described isolated tissue from cell (than specification cell). In addition can revise when calculating more specifically information and provide the most relatively on Average expression level, and Information base after computation Expression values.The expression values obtained in specification cell can form a kind of method and carry out the classification of severity to cancerous condition.
It is by preparing genomic DNA or mRNA/cDNA from the cell of an object samples (as turned in another embodiment Record polynucleotide), and described genomic DNA or mRNA/cDNA are compareed multi-nucleotide hybrid with one assess a labelling Expression, the most individual complete polynucleotide of these polynucleotide, it contains described labelling and labeled fragment.Many with comparison Before amplification oligonucleotide, it is possible to use arbitrarily polymerase chain reaction optionally expands cDNA.The expression of one or more labellings Quantitative PCR (QPCR) can be used to assess the expression of this labelling.
In a relevant embodiment, by the polynucleotide transcription mix obtained in sample and a substrate contact, this substrate In secure complete polynucleotide in a labelling of the present invention or at least some of (such as at least 7,10,15,
20,25,30,40,50,100,500 or more nucleotide residues).If one in a labelling of the present invention complete Whole polynucleotide or can find on substrate at least partially difference (as used different chromosomes or fluorescence to detect, Or it is fixed to different selectivity positions), then the expression of several labellings can use a single substrate simultaneously It is estimated (" gene chip " micro-matrixs of the polynucleotide being such as fixed on selectivity position).If using one to comment The method estimating marker expression level, it includes hybridizing a nucleotide with another, then described hybridization can be the most miscellaneous Complete under the conditions of friendship.
In another embodiment, it is possible to use the method for a compositions assesses the expression of a labelling.
One or more labellings of the present invention are detected owing to compositions of the present invention, test kit and method are to rely on Expression or the difference of copy number, in certain embodiments, the expression of described labelling or copy number can be significantly greater than described The minimum detection lowest limit of method, wherein said method be for assess the expression at least one specification cell and cancer cell or Copy number.
3. the method assessing expressing protein
The activity of one labelled protein or level can also by detection or quantitatively its polypeptide expressed detect and/or quantitatively.Described Polypeptide can detect and quantitatively by any method known to a person of ordinary skill in the art.These can include such as electrophoresis, Capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), cross diffusion chromatography etc. and analyze biological method, Or as liquid or gel precipitation react, immunity (single or double), immunoelectrophoresis, radio immunoassay (RIA) method, enzyme connection is exempted from Epidemic disease analytic process (ELISA kit), immune detection analytic process, immunoblotting, the various immunologys such as immunohistochemical method Method.Known albumen/antibody testing method is used for detecting whether cell have expressed a labelling of the present invention by one technical staff.
Another kind of reagent for detecting a peptide species of the present invention is a kind of antibody, and it can be combined with a peptide species, should Polypeptide is corresponding with the present invention labelling, such as a kind of antibody with a detectable label.These antibody can with polyclone or Monoclonal.One complete antibody or its fragment (such as Fab or F (ab') 2) can use.Relevant to probe or antibody Term " labelling " refers to by another reagent reacting with direct labelling, identically with direct label probe or antibody by one Can be connected (such as physical connection) with probe or antibody by detection substrate, make probe or antibody by direct labelling.The directly example of labelling Including using one primary antibody of a fluoroscopic examination, this fluorescent labeling secondary antibody label and the DNA of a band biotin The end labelling of probe, therefore it can detect with fluorescently-labeled streptavidin.
In another embodiment, described antibody marked, such as a radio label, chromosomal marker, fluorescence mark Note or the antibody of enzyme labelling.In another embodiment, antibody derivatives (such as an antibody and a Binding Capacity, or Be combined { such as biotin streptavidin } with albumen or the ligand of a protein ligand centering), or with an antibody sheet Section combines (such as a single-chain antibody, a single antibody height saltation zone), the egg that described fragment can be corresponding with a labelling White specific bond, if albumen is by the open reading fragment coding corresponding with labelling, or this albumen has at all or part of warp Later used after transcribing modification.
Immunohistochemistry or IHC refer to the principle that the antigen-specific utilizing antigen in biological tissue is combined, by antigen (such as albumen) It is positioned at the method in the cell of a tissue part.Immunohistochemistry coloring can make widely in paracytic diagnosis With, such as can find in cancer.Specific molecular marker is the feature of special porosity, as propagation or cell death (wither Die).IHC can also be widely used for studying the different piece understanding biomarker and the different albumen expressed a biological tissue In distribution and location.A kind of antibody antigen of imagination interacts and can realize in many ways.In the most frequently used example, one Plant anti-to be combined with a kind of enzyme, such as peroxidase, a kind of reaction generating color products can be catalyzed.Or this antibody also may be used With with a fluorogen labelling, such as fluorescein, rhodamine, DyLight Fluor or Alexa Fluor.
Albumen in cell can use technology known to a person of ordinary skill in the art to separate.The Protein Separation rule of this use is such as Be disclosed in those Harlow and Lane (Harlow and Lane, 1988, antibody: a kind of experiment guide, Cold Spring Harbor Laboratory Room publishing house, Cold SpringHarbor, New York).
In an example, these antibody or anti-fragment can use, such as western blot method or immunity in various methods Fluorescent technique detects the albumen of expression.In these use, can be by antibody or proteopexy in a solid support.Close Suitable solid phase support or carrier include any can be with an antigen or the support of an antibodies.Known support or carrier bag Include glass, polystyrene, nylon, polypropylene, polyethylene, glucosan, amylase, natural and modified cellulose, polypropylene Amide, gabbro and magnetic iron ore.
Other suitable carriers can being combined with antibody or antigen many known in those of ordinary skill in the art, it is possible to pin this A little supports use in the present invention.Such as, the albumen separated in cell can run a kind of polyacrylamide gel, and fixes In a solid phase support, such as NC Nitroncellulose.Then described support can be used after processing with detectable traget antibody again Suitably buffer rinsing.Then can be by the solid phase support twice described in buffer rinsing to remove unconjugated antibody.Solid phase The labelled amount combined in support then can detect by existing method.Using electrophoretic techniques detection method of protein is this Known to the those of ordinary skill of field (referring generally to R.Scopes (1982) Protein Purification,
Springer-Verlag,N.Y.;Deutscher,(1990)Methods in Enzymology Vol.182:Guide to Protein Purification,Academic Press,Inc.,N.Y.)。
In another embodiment, whether western blot (immunoblotting) is analyzed to may be used for detecting in sample has one many Peptide and content thereof.This technology generally includes and utilizes the gel electrophoresis carried out according to molecular mass to make sample Separation of Proteins out, (such as a kind of cellulose acetate membrane, a kind of nylon filter in the suitable solid phase support of Protein transfer to that will separate Or a kind of derivative nylon filter), described antibody to be hatched in sample, described antibody combines with a kind of polypeptide ground. Described anti-peptide antibody combines with the polypeptide ground in solid phase support.These antibody can use these labellings antibody (as Labelling goat anti-human antibody) carry out direct labelling or optionally by whole detection, described traget antibody can be with anti-polypeptide Ground combines.
In another embodiment, it is possible to use a kind of immunoassay detect polypeptide.As described herein, a kind of immunoassay Being a kind of assessment, it utilizes a kind of antibody to produce particular combination with analyte.Therefore to special between a peptide species and a kind of antibody Fixed combination detects, and this detection can characterize described immunoassay, and this is different from separation, targeting or quantifying analytes Other physically or chemically characteristic.
To the detection of described polypeptide and/or be quantitatively that the immunity using any one well to identify combines assessment (for example, see the U.S. is special Profit 4,366,241(incorporated by reference)；4,376,110(incorporated by reference)；4,517,288(incorporated by reference)；With 4,837,168 (incorporated by reference).Understand common immunity and may refer to Asai (1993) Methods in Cell Biology Volume37: Antibodies in Cell Biology,Academic Press,Inc.New York;Stites&Terr(1991)Basic and Clinical Immunology7th Edition。
Immunity combines assessment (or immunoassay) and generally uses one " agent for capturing " to carry out bound analyte being normally fixed specifically Analyte (polypeptide or sequence).Described agent for capturing be one can be with the composition of analyte specific bond.In another embodiment, Described agent for capturing is a kind of antigen, and it can combine a peptide species specifically.Described antibody (polypeptide) can be by any one Prepared by method known to a person of ordinary skill in the art.
Immunoassay generally also can use a kind of marking agent to combine specifically and combination complex described in labelling, and it is by catching Catch what agent and analyte were formed.Described marking agent self can be a kind of composition including antibody/antigen complex.Therefore, institute The marking agent stated can be polypeptide or the antibody of a kind of labelling of a kind of labelling.Or described marking agent can be one the three one-tenth Point, such as another kind of antibody, it can be combined specifically with described antibody/polypeptide complex.
In one embodiment, described marking agent is the second human antibody of a kind of tape label.Or described second antibody can To lack a labelling, but it can transfer by the 3rd antibodies of a labelling, and the 3rd described antibody is specific to second The derivative extraordinary antibody of antibody.Described second antibody can be to modify with detectable composition, such as biotin, so It just can be combined, such as the streptavidin of enzyme labelling by a kind of 3rd labelling molecular specific.
Other can also be as marking agent with the albumen of the constant series specific bond of immunoglobulin, such as protein A or Protein G Use.These albumen are the common constituents of streptococcal cell wall.They produce with the immunoglobulin constant series of various special types A kind of strong immunogen reaction (referring generally to Kronval, et al. (1973) J.Immunol., 111:1401-1406, And Akerstrom (1985) J.Immunol., 135:2589-2542).
As it has been described above, the immunoassay of detection and/or a quantitative peptide species can use known to a person of ordinary skill in the art various Method.
The preferred immunoassay detecting a peptide species can be competitive or noncompetitive.Non-competitive immunoassay can be assessed The amount of the seizure analyte that can directly detect.Such as in one " sandwich " is assessed, described agent for capturing (anti-peptide antibody) Can with a solid substrate directly in conjunction with, and be fixed on substrate.Then these fixing antibody can catch in test sample Polypeptide.Therefore described immobilized polypeptide is then combined with a marking agent, such as the second human antibody of a kind of tape label.
In competitive evaluation, analyte (polypeptide) amount contained in sample is to add (external source) analyte by detection one The amount of (polypeptide) directly detects, and this interpolation analyte can from a kind of agent for capturing, (anti-peptide resists by analyte contained in sample Body) middle replacement out (or competition).In a competitive evaluation, the polypeptide of a kind of known quantities can join described In sample, make to connect this and contact with a kind of agent for capturing.The peptide concentration contained with sample with the polypeptide amount of antibodies is inversely proportional to.
In another embodiment, described antibody is integrally fixed in a solid substrate.Measure in one peptide species/antibody complex The non-complex polypeptide amount of contained polypeptide amount or optionally measurement residual may determine that the polypeptide amount with antibodies.Polypeptide amount Can detect by arranging the polypeptide of a labelling.
Assessment of the present invention can use standard method known to a person of ordinary skill in the art to complete the (sun as polypeptide Property is negative or quantitative).The ad hoc approach completed is completed by evaluation form and selected marker.Such as, enzyme labelling is formed Color products may be used for realizing a kind of immunoblotting assay.When exist a kind of apparent trace or combine can produce feminine gender During result, the apparent coloured combination of one of correct molecular weight or trace can produce positive findings.Strong combination or print Mark can detect polypeptide quantitatively.
The antibody used in various immunoassay of the present invention can method be prepared as described in the present invention.
In another embodiment, level (active) is to be lived by the enzyme of measurement gene outcome and assess out.Assessment one The method that enzyme is lived is known to a person of ordinary skill in the art.
The in-situ techniques of one labelled protein of detection includes the traget antibody introducing an anti-Protein Detection in an object.Such as, Described antibody can be with on labelling radioactive label, and its existence is positioned in an object, can come with standard imaging techniques Detection.
Some labellings identified by method of the present invention can be secretory protein.Technical staff can determine any specific simply Whether labelled protein is a kind of secretory protein.Determining to realize this, labelled protein is at a mammalian cell, such as one Expressing in human cell line, collect extracellular fluid, whether assessment extracellular fluid contains described albumen (such as uses an energy and egg The traget antibody of white specific bond).
A kind of method example described below may be used for detecting a kind of protein excretion.About 8x105293T cell can be Growth medium (Dulbecco improve Eagle's medium DMEM} its added with 10% hyclone) cultivate, condition of culture is 37 °, being passed through 5% (v/v) CO2,95% air enters in about 60-70% mixture.Then a kind of Standard transfection mixing is used Cell described in thing transfection, contains the DNA and the LipofectAMINE of 10 microlitres of 2 micrograms in the mixture in the most each holeTM (GIBCO/BRL catalog number (Cat.No.) 18342-012), wherein said DNA has the expression vector of a kind of encoding proteins.Described turns Dye mixture is placed about 5 hours, is then changed to fresh growth medium, and places in atmosphere.Gently rinse with DMEM Twice of each hole, does not wherein contain methionine or cysteine (DMEM-MC in DMEM;ICN catalog number (Cat.No.) 16-424-54).Again DMEM-MC and about 50 micromicrocuries Trans-of about 1 milliliter are added in each hole35STMReactant (ICN catalog number (Cat.No.) 51006). It is passed through 5%CO to this some holes2Gas, and cultivate a selective time at 37 DEG C.Then remove in cultivation subsequently The conditioned medium of 150 microlitres, and centrifugal segregation floated cells and fragment.Albumen on floating on the surface i.e. illustrates that albumen is secreted.
It should be appreciated that these object samples, as one is organized, whole blood, serum containing expectorant, bronchoalveolar lavage, hydrothorax, Blood plasma, oral cavity scrapes, saliva, cerebrospinal fluid, urine, stool, can contain cell in the sample of bone marrow, especially thin when these Born of the same parents can be cancer, is especially metastatic when cancer, therefore can use in method of the present invention.In assessment Before marker expression level in described sample, it is possible to use various known after collect preparation and storing technology (as nucleotide and/ Or protein extraction, fixing, store, freezing, ultrafiltration, concentrate, evaporation, centrifugal etc.) process described cell sample.Cause This compositions of the present invention, test kit and method may be used for detection with protein to marker expression just, these protein In a kind of be positioned at its express cell surface.Technical staff can determine that the albumen corresponding with any specific labelling is simply No include a cell surface protein.Such as immunological method may be used for these albumen detecting in whole cell, or public The sequence analysis that the computer known supports is (such as SIGNALP program；Nielsen et al.,1997,Protein Engineering 10:1-6) may be used for predicting that the existence of at least one extracellular domain (such as includes two kinds of secretory proteins, at least one, these albumen Extracellular domain).The expression of one labelling just can be able to detect without cell lysis, and a protein corresponding with this labelling is at least Some surface being in its cell expressed (such as uses the antibody of a kind of labelling, it and one of described albumen Extracellular domain combines specifically).
Present invention additionally comprises test kit, they can detect a biological specimen, as one containing expectorant, and bronchoalveolar lavage, hydrothorax, Tissue, whole blood, serum, blood plasma, oral cavity scrapes, saliva, cerebrospinal fluid, urine, stool, whether the sample of bone marrow contains with A polypeptide that labelling of the present invention is corresponding or nucleotide.These test kits are determined for whether an object suffers from cancer or tool Suffer from cancered risk.Example test kit as mentioned can include compound or the reagent of a labelling, and it is many that it can detect one Peptide or mRNA, this peptide species or mRNA are the polypeptide that coding is corresponding with labelling of the present invention in a biological specimen, also wrap Include and determine that the composition of the polypeptide contained by sample or mRNA amount is (such as a kind of antibody being combined with polypeptide or a kind of and coded polypeptide DNA Or the oligonucleotide probe of mRNA combination).
Such as the test kit of antibody substrate, described test kit includes: (1) first antibody (is such as attached to a solid phase prop up In support), it is combined with polypeptide, and described polypeptide is corresponding with a kind of labelling of the present invention；And selective (2) a kind of second Different antibodies, it is combined with polypeptide or first antibody, and is conjugated with a detectable labelling.
Such as the test kit of antibody substrate, described test kit includes: (1) oligonucleotide, such as a detectable label Oligonucleotide, it is with a nucleotide sequence hybridization, this nucleotide sequence coded one corresponding with labelling of the present invention Polypeptide or (2) a pair primer for one nucleic acid molecule of amplification, wherein said nucleic acid molecule and of the present invention Individual labelling is corresponding.This test kit can also include, such as a kind of buffer agent, a kind of preservative or a kind of protein stabiliser.Described Test kit also includes the neccessary composition of those detection detectable labels (such as a kind of enzyme or substrate).This test kit is possibly together with one control Sample preparation this or a series of control sample, they can be evaluated and contrast with test sample.Each composition in test kit can be used One independent container encapsulates, and puts into one individually together with the description of all of container explanation result needed for using test kit Packaging in.
4. the method that evaluation structure changes
Present invention additionally comprises one and assessed whether a structural change, such as the method for sudden change.
Another kind of detection method is probe and allele specific hybridization, and it uses the probe overlapping with polymorphic site, and in institute About 5,10,20,25 or 30 nucleotide are had around the polymorphic district stated.In an alternative embodiment of the invention, can be with sudden change The various probes of specific hybrid are affixed in a solid phase support, such as one " chip ".Oligonucleotide can in many ways, To be combined with a kind of solid phase support including lithography.Such as can there be 250,000 oligonucleotide (genes on one chip Chip, AffymetrixTM).Such as Cronin et al. (1996) Human Mutation7:244 discloses abrupt climatic change Analyze, it use these chips including oligonucleotide, also referred to as " DNA probe matrix.In one embodiment, One chip includes all sudden changes at least one gene polymorphic district.Then described solid phase support connects with a test oligonucleotide Touch, and with specific probe hybridization to be detected.Therefore, on one or more genes, the density of multiple sudden changes can be with a sample This hybrid experiment is identified.Such as can identify the nucleotide polymorphism in the controlling elements of 5' upstream with a single hybrid experiment Sudden change.
In other detection method, needed first to expand at least some of labelling before identifying sudden change.Amplification can use ability Method known to territory, such as, be shown in Wu and Wallace (1989) Genomics4:560 with PCR and/or LCR() realize. In one embodiment, the genome of a cell can be cracked into two PCR primer, and expands many circulations until its product Amount can reach DNA amplification need amount.In certain embodiments, these primers are between 150 and 350 base pairs.
Selective amplification method includes: certainly maintain sequence replicating (Guatelli, J.C.et al., (1990) Proc.Natl.Acad. Sci.USA87:1874-1878), Q-β replicative enzyme (Lizardi, P.M.et al., (1988) Bio/Technology6:1197) Certainly sequence replicating (Guatelli et al., (1989) Proc.Nat.Acad.Sci.87:1874) and nucleoside soda acid are maintained Basic sequence replicates (NABSA) or other any amplification oligonucleotide method, subsequently by using technology known to a person of ordinary skill in the art Detect amplifier molecule.These detection schemes are particularly useful for detecting the nucleic acid molecule of those low contents.
In one embodiment, any sequencing reaction well known in the art can be directly used in the order-checking of at least some of labelling,
And by sample sequence and corresponding relevant (control) alignment, detection sudden change.Preferably sequencing reaction includes that those are based on U.S. Add clean and technology (Proc.Natl Acad Sci USA (1977) 74:560) or Sanger (Sanger of gilbert's improvement et al.(1977)Proc.Nat.Acad.Sci74:5463).Arbitrarily row can also be used journey is filled when carrying out object evaluation Sequence (Biotechniques (1995) 19:448), including with the order-checking of quantitative spectra algoscopy (for example, see United States Patent (USP) 5,547,835 (incorporated by reference)) and H.Publication No. WO94/16101 of application, entitled passes through exonuclease The quantitative spectra algoscopy of degraded carries out the international patent application (incorporated by reference) of DNA sequencing, and United States Patent (USP) 5,605,798 (is drawn With reference) and H.Application No. PCT/US96/03651, entitled DNA based on quantitative spectra algoscopy diagnose International application (incorporated by reference)；Cohen et al.(1996)Adv Chromatogr36:127-162;and Griffin et al.(1993)Appl Biochem Biotechnol38:147-159).In certain embodiments, the common skill of this area Art personnel can be appreciated that only one, and two or three nucleotide bases need to determine in sequencing reaction.Such as, A-track etc. are only One nucleotide of detection can realize.
The United States Patent (USP) 5,580,732 of entitled " the DNA sequencing method of the DNA polymer chain type probe using to mix " United States Patent (USP) 5,571,676(of (incorporated by reference) and entitled " method of dislocation in detection DNA sequencing in situ " quotes ginseng Examine).
In some instances, limit enzyme analyte and can demonstrate the unique allele of labelling in object DNA.Such as, a spy Specific nucleotide is polymorphic can be formed in a nucleotide sequence, and it includes a restriction site, and due to another nucleotide The sudden change of sequence and lack.
In a preferred embodiment, the protection of decomposition agent (such as a kind of nucleotide, azanol or Osmic acid. and piperidines) can For base mismatch (Myers, the et al. (1985) produced in detection RNA/RNA DNA/DNA or RNA/DNA heteroduplex Science230:1242)." mismatch cleavage " technology starts typically by a kind of heteroduplex controlling nucleotide formation of hybridization , described control nucleotide can optionally be labeled, and as by RNA or DNA marker, it includes a labelling sudden change Nucleotide sequence, this sequence has the sample nucleotide obtained from a tissue samples, such as RNA or DNA.Described is double Chain can be by a kind of agent treated, and then double-strand breakage becomes strand, if double-strand is to control the base-pair mismatch shape between sample chain Become.Such as, RNA/DNA double-strand can be with RNase process, and DNA/DNA hybrid is processed as decomposing mistake by S1 nucleotidase Join the enzyme in district.In further embodiments, DNA/DNA or RNA/DNA double-strand can process with azanol or Osmic acid., and Mispairing district is decomposed with sending pyridine to process.After mispairing district is decomposed, the material of generation is then coated with denaturing polyacrylamide gel, Determine whether control and sample nucleotide have a qualification nucleotide sequence or nucleotide therein the most different, then separated. For example, see Cotton et al (1988) Proc.Natl Acad Sci USA85:4397;Saleeba et al(1992) Methods Enzymol.217:286-295.In another embodiment, described control or sample nucleotide have been by using Labelling in detection.
In another embodiment, can with high energy liquid chromatograph (DHPLC) identify a kind of sudden change (Oefner and Underhill, (1995)Am.J.Human Gen.57:Suppl.A266).DHPLC uses reverse-phase paired ion chromatography to detect heteroduplex, These heteroduplexs are in PCR fragment expands, and are produced by with a specific nucleotide site on the hybridization monomer of this fragment (Oefner and Underhill(1995)Am.J.Human Gen.57:Suppl.A266).PCR primer is typically to make It is connected to what handled DNA both sides produced by PCR primer.DHPLC has analyzed, and then analyzes produced chromatograph, root Identify base pair change according to specific chromatogram or deletion (sees O ' Donovan et al. (1998) Genomics 52:44-49)。
In other embodiments, the change of electrophoretic mobility can be used for differentiating the type of labelling sudden change.Such as single strand conformation poly morphism (SSCP) may be used for current transfer rate difference (Orita et al. (1989) Proc between detection sudden change and various nucleotide Natl.Acad.Sci USA86:2766,see also Cotton(1993)Mutat Res285:125-144;and Hayashi (1992)Genet Anal Tech Appl9:73-79).The Single-stranded DNA fragments of sample and control nucleotide can be with degeneration with multiple Property.Second structure of single-stranded nucleotide can change according to sequence, and the change of electrophoretic mobility even makes an independent base Detection change.DNA fragmentation can be with labelling or detection labelling.Use RNA(rather than DNA) can improve assessment quick Sensitivity, wherein the second structure is more sensitive to the change in sequence.In another embodiment, described object method uses hybridization Analyze and separate hybrid double-stranded molecule (Keen et al. (1991) Trends Genet7:5) according to the change of electrophoretic mobility.
In another embodiment, the movement analyzing a nucleotide can identify the sudden change in a polymorphic district, including polypropylene Polymorphic district in acrylamide gel, it contains a conformational polymerphism (DGGE) using conformational polymerphism gel electrophoresis to assess (Myers et al.(1985)Nature313:495).When using DGGE as the method for analysis, DNA can be modified Ensure it will not degeneration completely, such as utilize PCR add height dissolve GC-concentration of DNA one about 40bp GC.Enter at one In the embodiment of one step, a variations in temperature is substituted for the change of denaturant, identifies the control after modification and sample DNA Difference (Rosenbaum and Reissner (1987) Biophys Chem265:1275).
Detect that technical examples that two nucleotide produce at least one nucleotide difference is nonrestrictive includes that selectivity oligonucleotide is miscellaneous Hand over, selective amplification or selectivity primer extension.Such as oligonucleotide probe is intensively to replace known polymorphism core when preparing Thuja acid (allele-specific probe), then hybridizes with target dna under the hybridization permissive condition only finding a best pairing (Saiki et al.(1986)Nature324:163);Saiki et al(1989)Proc.Natl Acad.Sci USA 86:6230;and Wallace et al.(1979)Nucl.Acids Res.6:3543).This allele-specific is few Hybridization techniques may be used for simultaneously detecting the various nucleotide change occurred in the polymorphic district of difference of labelling.The most such as Really oligonucleotide has the nucleotide sequence of specific mutations, then it is attached on a hybridization thin film, then this thin film and mark The sample nucleotide hybridization of note.Then analyze hybridization signal and can obtain the detection of the nucleotide to sample nucleotide.
Or the allele specific PCR according to selective PCR amplification can use in an embodiment of the present invention.Few Nucleotide can as primer under suitable conditions, in the molecule between or at the 3' end of a primer, specifically expand and need The sudden change of amplification, so can prevent mispairing or reduce the extension (therefore amplification is to carry out according to different hybridization) of polymerase (Gibbs et al(1989)Nucleic Acids Res.17:2437-2448).This technology is referred to as " PROBE ", It is for the extension of probe oligonucleotides base.In addition it has also drawn a new restriction site at saltation zone, thus is formed Base cracking detection (Gasparini et al (1992) Mol.Cell Probes6:1).
In another embodiment, United States Patent (USP) 4,998,617 and Landegren, U.et al., (1988) Science 241:1077-1080 discloses a kind of oligonucleotide connection assessment (OLA) of use and identifies sudden change.Described OLA scheme uses two Planting oligonucleotide, they can be with an adjacent independent object chain sequence hybridization.A kind of oligonucleotide and a single labelling, As biotinylated labelling connects, another is detectably labeled.If being found that exact complementarity in a target molecule Sequence, then these oligonucleotide can hybridize, and therefore their position is adjacent, formed one connect substrate.Then connecting can To use avidin or other biotin ligand to carry out the oligonucleotide of overlay marks.Nickerson, D.A. etc. are open A kind of nucleotide check and evaluation, the characteristic of PCR and OLA combines (Nickerson, D.A.et al., (1990) by it Proc.Natl.Acad.Sci.(U.S.A.)87:8923-8927).In this approach, PCR has been used for target dna Exponential amplification, and described target dna can use OLA to detect.
Present invention also offers the method detecting independent nucleotide polymorphisms in a labelling.Owing to same sequence area can be used Expanding in the both sides of various independent nucleotide polymorphic site, therefore their analysis does not requires to identify that single nucleotide is present in On various sites, it is not also required to determine a complete gene order for each object.The method of various improvement can convenient this The analysis of a little individually nucleotide polymorphisms.
In one embodiment, single nucleotide polymorphisms can use a specific anti-exonuclease, such as such as Mundy, C.R. described (United States Patent (USP) 4,656,127 (incorporated by reference)).According to described method, a primer and described location Gene order 3' immediately is complementary, and therefore pleomorphism site can be miscellaneous with a target molecule obtained from particular animals or human body Hand over.If the pleomorphism site on target molecule contains a nucleotide, it and existing specific anti-Exonucleolytic enzyme derivative Complementation, then derivant can be connected with the end of hybridized primer.These connections make primer be resistant to excision enzyme, and it is right therefore to realize Its detection.Although the qualification example of anti-Exonucleolytic enzyme derivative is known, but find that this primer of deuteron can become Become anti-excision enzyme to invert, the pleomorphism site of target molecule exists protokaryon thuja acid mutual with the nucleotide derivative of use in reaction Mend.The advantage of this method is that it need not determine the information of a large amount of exogenous array.
In another embodiment of the present invention, a kind of method based on solution is the nucleotide for identifying a kind of pleomorphism site (Cohen,D.et al.French Patent2,650,840；PCT Publication number: WO91/02087 (incorporated by reference)).As United States Patent (USP) 4,656, described in the Mang Difa described in 127 (incorporated by reference), the primer of use is mutual with gene location sequence 3' immediately Benefit becomes a pleomorphism site.Described method can use the dideoxyribonucleoside acid derivative of labelling to identify the nucleoside in this site Acid, if its nucleotide complementary with polymorphic site, then just could be connected with the end of primer.
Goelet, P. etc. (PCT Application No. 92/15712 (incorporated by reference)) disclose a kind of process for selective, i.e. gene imprinting Analytic process or GBA.This method of Goelet, P. etc. uses end and the mixture of a primer of labelling, wherein primer with Sequence 3' be complementarily shaped to a kind of pleomorphism site.Described labelling end is connected, hence with described core complementary therewith Thuja acid detects, and this nucleotide is present in the polymorphic site of assessed target molecule.Method (French with Cohen etc. Patent2,650,840;PCT Publication number: WO91/02087 (incorporated by reference)) to compare, the method for Goelet, P. etc. is one Planting hybridization to assess mutually, wherein said primer or target molecule are integrally fixed in a solid phase.
For assessing the various guiding prime nucleotide linkers of polymorphic site in DNA, (Komher, J.S.et are disclosed al.,(1989)Nucl.Acids.Res.17:7779-7784;Sokolov,B.P.,(1990)Nucl.Acids Res. 18:3671;Syvanen,A.-C.,et al.,(1990)Genomics8:684-692;Kuppuswamy,M.N.et al.,(1991)Proc.Natl.Acad.Sci.(U.S.A.)88:1143-1147;Prezant,T.R.et al.,(1992) Hum.Mutat.1:159-164;Ugozzoli,L.et al.,(1992)GATA9:107-112;Nyren,P.(1993) et al.,Anal.Biochem.208:171-175).These methods and GBA difference are that they are all linkage flags Deoxydization nucleotide distinguishes multiple bases of a pleomorphism site.Although so signal becomes ratio with the Deoxydization nucleotide quantity being connected Example, but run the polymorphism that identical nucleotide occurred and can produce signal (Syvanen, A.C., the et proportional to length of running al.,(1993)Amer.J.Hum.Genet.52:46-59)。
In order to differentiate the sudden change of a polymorphic sequence being positioned at a label coding district, it is also possible to use different from said method Method.Such as, using a kind of antibody can identify the sudden change of one mutation markers of a kind of coding, described antibody can be known specifically The albumen not suddenlyd change, as in immunohistochemistry or immunity.Known method can be used to prepare antibody to wild type marker Or saltant type labelling.
Or the activity of a labelling can also be measured, as being combined with a tagged-ligand.It is to it is known in the art that in conjunction with assessment Including such as obtaining cell from a subject, complete Binding experiment by the ligand of a labelling, determine the knot with mutain Close and whether be different from the combination with wild-type protein.
VI. preferred screening method based on ALK inhibitor
Present invention also offers qualification substrate, suppression ALK polypeptide (such as EML4-ALK polypeptide), thus suppress cancer cell proliferation, Growth, sudden change, apoptosis and/or the method for transfer.These methods include testing compound and an ALK polypeptide (such as table by one Polypeptide listed by 1) contact.In certain embodiments, described ALK polypeptide includes a variant (as listed in table 1 polymorphic), It can increase and suppress risk that is imperfect or that be not responding to what one or more ALK inhibitor were formed.A kind of compound is a kind of swollen The inhibitor of tumor metastasis, just can identify this chemical combination according to a kind of compound of testing to the effect of the activity measurement of ALK polypeptide variants Thing (such as includes that ligand combines, as ATP combines and/or the activity of tyrosine kinase).In a specific embodiment, A kind of compound of testing can suppress the activity of tyrosine kinase, by this activity compared with the activity do not tested in the presence of compound Whether the most just can identify this test compound can be as the inhibitor of a kind of tyrosine kinase.If the compound of a kind of ALK presses down Agent activity changes, then it just can assess its suppression tumor growth or ability of transfer further.
Preferably activated tyrosine kinase sudden change includes the new labelling (as ALK suddenlys change) listed by table 1 of the present invention, and it can be used for reflecting Make for treating, improve or anticancer compound, such as, draw or prevent cancer cell multiplication, growth, sudden change, apoptosis and/or turn Move.Can control, such as suppression, neutralize, intensify or the screening chemical libraries of model molecule is well known in the art.Described chemical libraries Can be such as peptide storehouse, peptidomimetic class libraries, chemically synthesized library, restructuring, as antibiotics display storehouse and original position substrate transcribe storehouse, other Non-peptide synthesis of organic substance storehouse.
Screening or formed, identifies, selects the suitable high homology inhibitor of neoplasm labelling listed by table 1 of the present invention (as ALK is prominent Become) can realize by various methods.Putting it briefly, they include two class schemes without limitation.One class is to use target protein Structure knowledge designs a preferred molecule that can accurately act on.One example is computer aided molecular design, is based especially on New structure function information shown in Fig. 6.Another kind of is to use combination or other molecular library, and therefore a macromole storehouse is permissible Screened for affine relevant target enzyme, or the activity of target enzyme can be suppressed.In one further embodiment, may be used The antibody of target enzyme can be suppressed screening a plate.
Embodiments more of the present invention include determining the neoplasm labelling that a specific compound can suppress listed by table 1 of the present invention (as ALK suddenlys change).The activity that can compare test compound and be widely known for treating the compound of neoplastic lesion, assesses Test compound directly or indirectly treats the ability of neoplastic lesion.Such as, test compound suppresses new listed by anti-table 1 of the present invention The ligand of biomarker combines, and combines such as ATP and/or the ability of tyrosine kinase activity (as ALK suddenlys change) can be with known ALK inhibitor, as PF-02341066 and/or PDD compares.In one embodiment, under same evaluation condition, these Test compound the inhibitory action of neoplasm labelling (as ALK suddenlys change) listed in table 1 of the present invention is at least known in ALK press down The 99.6% of preparation, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98.5%, 98%, 97.5%, 97%, 96.5%, 96%、95.5%、94%、93.5%、93%、92.5%、92%、91.5%、91%、90.5%、90%、89.5%、89%、88.5%、 88%、87.5%、87%、86.5%、86%、85.5%、85%、84.5%、84%、83.5%、83%、82.5%、82%、81.5%、 81%、80.5%、80%、79%、78%、77%、76%、75%、74%、73%、72%、71%、70%、69%、68%、67%、 66%、65%、64%、63%、62%、61%、60%、59%、58%、57%、56%、55%、54%、53%、52%、51%、 50% or within the scope of these.In certain embodiments, cell can transfect a structure, and this structure can code book invention table 1 Listed neoplasm labelling (as ALK suddenlys change), and contact with a test compound, this compound indicates one and can detect Labelling, then analyse whether exist combine test compound.In certain embodiments, described transfectional cell can be with test Compound combines, and with those, these cells have been transfected the thin of neoplasm labelling listed by table 1 of the present invention (such as ALK sudden change) Born of the same parents compare, and illustrate that testing compound combines with the neoplasm labelling (as ALK suddenlys change) listed by table 1 of the present invention, therein Biomarker is expressed by these cells.This combination of described compound is typically by any one assessment well known in the art Determine, as ELISA, RIA and BIAcore assess.
Use a kind of recombinant expressed sudden change of described enzyme or from a homolog of another species isolated or ortholog body Can be according to compound to neoplasm labelling (as ALK suddenlys change) active suppression listed in table 1 of the present invention or other effect Screen these compounds.Or the cell of these neoplasm labeling polypeptides is expressed can be by one test compound treatment, one Test compound effect in the phosphorylation of species specificity target can be determined, such as, use a kind of technology of the present invention. In one embodiment, tyrosine kinase activity is determined.Determining that tyrosine kinase affects the method (such as suppression) of activity is this Known to the those of ordinary skill of field.In certain embodiments, tyrosine kinase activity can also be by assessing the phosphorus of a labelling Hydrochlorate (such as the phosphate of 32P-labelling) determines with the combination of a substrate, and wherein said substrate can use table 1 institute of the present invention The neoplasm labelling (as ALK suddenlys change) of row carrys out phosphorylation (as a part or a fragments of peptides, especially those dirty signals become Point).In other embodiments, tyrosine kinase activity can use a kind of common tyrosine kinase activity test kit to detect (example Such as common tyrosine-kinase enzyme detection kit (Takara Bio, Inc., Madison, Wis.)；Tyrosine kinase detection examination Agent box (Millipore, Billerica, Mass.))
In another embodiment, described screening technique also includes determining the life whether described compound slow down tumor cell Long, such as known tumor cell can express the tyrosine-kinase enzyme mutant of a kind of activation, a kind of neoplasm as listed by table 1 of the present invention Labelling (as ALK suddenlys change).Can use various cell line, selection is that those of ordinary skill in the art is public according to its tissue Know (such as the BA/F3 cell) that can detect.The most various cell lines are well characterized, and are ground by such as National Cancer Study carefully in the screening sequence of the new type anticancer medicine that institute (NCI) is used in its research and development.
Efficiently and notable efficient growth of tumour cell inhibitor, as at 100 μMs, 90 μMs, 80 μMs, 70 μMs, 60 μMs, 50μM,40μM,30μM,20μM,10μM,9μM,8μM,7μM,6μM,5μM,4.5μM, 4μM,3.5μM,3μM,2.5μM,2μM,1.5μM,1μM,900nM,850nM,800nM,750 nM,700nM,650nM,600nM,550nM,500nM,450nM,400nM,350nM,300nM,250nM, 200nM,150nM,100nM,95nM,90nM,85nM,80nM,75nM,70nM,65nM,60nM,55 nM,50nM,45nM,40nM,35nM,30nM,25nM,20nM,15nM,10nM,5nM,4nM,3nM, When 2nM, 1nM or lower dosage, effective percentage is more than 50%, and it also illustrates that described compound can be effectively used to treat tumprigenicity Pathological changes.A kind of IC50Value may determine that and for contrasting.This value is raw relative to controlling required suppression tumor cell by 50% Long drug level.
These values can also use other standard.The most in further embodiments, screening method of the present invention also includes determining Whether test compound can cause the tumor cell generation apoptosis in cultivation.Two kinds of multi-forms of cell death can use morphology Describe with microbial administration: downright bad and apoptosis.The permeability increasing plasma foil can produce necrosis, therefore cell therewith Expand, and plasma foil in seconds ruptures.Thin film bubbles, Cytoplasm condensation and the activity of endogenous endonuclease It it is the feature of apoptosis.
Normal structure updates and organ, can naturally produce apoptosis during the embryonic development of extremity.Various stimulating factors are by ionization spoke Penetrate and also can apoptosis-induced produce with some chemotherapeutics, including cytotoxic T lymphocyte and natural killer cell.Apoptosis different Often controlling is to act as an important effect under including the various pathology such as cancer, AIDS or Alzheimer.
Culture of tumor cell under these conditions, the then induction of filler test compound on intracellular apoptosis.These screening techniques In some embodiments, before or after being included in the test compound of various concentration with test compound treated cells, merge culture medium also Process one to seven day.The cell of apoptosis can be measured in the attachment of culture medium or " floating " part.Both of which can be so Collect: remove floaters on surface, trypsinization attached cell, a centrifugal elutriation step (10 minutes, 2000rpm) it Prepared by rear combination two kinds.After one test compound treatment, culture medium can be assessed for apoptosis and necrosis, such as at mark Fluorescence microscope is used after having remembered acridine orange and ethidium bromide.The method of various measurement apoptotic cells is those of ordinary skill in the art Known, such as Duke&Cohen disclose a kind of measure apoptotic cell quantity method (Curr.Prot.Immuno., Coligan et al.,eds.,3.17.1-3.17.1,1992).Such as use trypsin and flush three times with PBS, receiving Collect the floating and cell of attachment.Then the aliquot of cell is centrifuged.Described granule is again suspended in medium, Yi Zhonghan The dye mixture having acridine orange and ethidium bromide is prepared from PBS, and is gently mixed.Then described mixture can To be fixed in a microscope chute, check the morphological characteristic of its apoptosis.In measurement cell, the increments of DNA fragmentation can also Detect apoptosis quantitatively, cell therein test compound treatment.Business luminosity enzyme immunoassay (EIA) (EIA) can be used Detect quantitatively cytoplasmic protein-relevant-DNA-fragment (monomer and oligonucleotide) (such as Cell Death Detection ELISA, Boehringer Mannheim)。
In a further embodiment, the screening method of the present invention also includes determining the transplanting whether test compound reduces tumor, as At an animal model transplanted.The method of assessment tumour transplatation is known to a person of ordinary skill in the art (for example, see Khanna and Hunter,Carcinogenesis26:513-523,2005).A kind of transplantation model includes people-Mus xenotransplantation, wherein Human cancer cell system or tissue can be transplanted in the mice of compromised immune wound (such as SCID mice or nude mouse).Similar Method in, cell line can be designed as a kind of new biomarker (as ALK suddenlys change) expressed listed by table 1 of the present invention, It can be transplanted in the mice of a kind of compromised immune wound.In one embodiment, tumor cell or cell line can be direct It is injected in system circulation.The site injected determines establiss site in these experimental systems largely.Experimental metastasis It is in mouse tail vein both sides that tumor cell the most frequently used in model injects site, and it is mainly generation in Lung metastases.Tumor is thin Spleen in born of the same parents is interior or hepatic portal vein injection is the most frequently used site cultivating transplanting in live body, and the intracardiac injection of cell can turn Shifting produces multiple site, including bone.After tumor cell or other cell line are injected into circulation, at target site (such as lung) The transfer of upper cultivation in some skies or can monitor after week.
The another kind of model of assessment neoplasm metastasis is to use Chang Weiyi to plant, and wherein cancer cell can be transplanted to normal position or tissue, in It is that a tumor is derivative (be such as directly injected into or surgery cultivates tumor fragment) from here.The transfer naturally often produced in the tumor of position Can assess in several days or a few week.A kind of test compound reduce or prevent neoplasm metastasis ability can by by one test It is injected in tumor cell to compound subcutaneous injection, intramuscular injection, blood circulation or orthotopic transplantation.The quantity that transfer is cultivated, Size or time can be assessed.A kind of compound suppressing neoplasm metastasis can reduce the quantity of transfer, such as ratio and control sample At least reduce 10%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or even100%.A kind of compound that can suppress neoplasm metastasis also can make the size of transfer, and to control sample little than one.Similarly, one Kind suppression neoplasm metastasis compound can also slow down transfer cultivate beginning, for example, at least one week, two weeks, one month, 6 Month, 1 year or the most indefinite.
VII. preferred ALK inhibitor
Method of the present invention includes identifying whether a kind of object can be with the new biomarker listed by table 1 of the present invention (such as ALK Sudden change) treat, inducing death of neoplastic cells, hinder tumor growth or reduce the risk of neoplasm metastasis.ALK peptide inhibitor It is well known in the art.Such as PF-02341066, PDD, 2-methyl isophthalic acid 1-(2-methyl-propyl)-4-oxygen-4,5,6,11,12,13 -hexahydro-2H-indazole [5,4-α] pyrroles [3,4-c] carbazole-8-base [4-(dimethylamino) benzyl] methyl carbamate, (1S, 2S, 3R, 4R)-3-({ the chloro-2-of 5-[(1-ethyl-2,3,4,5-tetrahydrochysene-6-methoxyl group-2-oxygen-1H-1-benzazepine-7- Base) amino]-4-pyrimidine } amino) dicyclo [2.2.1] hept-5-alkene-2-Methanamide, and NVP-TAE684, for example, see PNAS 104:270-275,2007;Choi,Y.L.et al.(2008)Cancer Res.68:4971-2976;And Biochemistry48:3600-3609,2009, they are incorporated by reference at this.
Incorporated by reference
Each independent publication, patent or patent application represent when including reference in especially and individually, of the present invention all Publication, patents and patent applications is all incorporated as reference.When they are conflicting, the application, including the present invention's It is defined and will be played a decisive role.
That is additionally correlated with in full with it in it is all included in reference to public database assesses the relevant any polynucleotide of number and polypeptide Sequence, the biological skill of those and/or the country such as kept on the WWW of tigr.org by Joint Genome Institute (TIGR) Those kept on the art information centre (NCBI) WWW at ncbi.nlm.nih.gov.
Example
The present invention is further described by example below, should not be construed as limiting.All contents related to, numeral, sequence List, patent and the patent document quoted belong to the present patent application scope.
Example 1-is used for raw material and the method for example 2-4
A.DNA checks order
Oligomerization (dT)-primer cDNAs uses EZ1 system (Qiagen, Valencia, CA) to extract also from sample RNAs With PrimeSTAR HS archaeal dna polymerase (Takara Bio Inc., Shiga, Japan) and primer ALK-TK-F
(5 '-TACAACCCCAACTACTGCTTTGCT-3 ') and ALK-TK-R1 (5 '-AGGCACTTTCTCTTCCTCTTCCAC-3 ') Obtained by polymerase chain reaction (PCR) 30 circulation (when including 98 DEG C when 10 seconds and 68 DEG C 2 minutes).PCR primer Identical with ALK kinase domain, then disperse and check order by Illumina genomic analysis system II (GAII), passing through It is 76 substrate that pairing end sequencing system (Illumina, San Diego, CA) obtains two ends.Reading initial data is to exist PCR original order and quality >=20 based on all substrate of quality-infiltration.Penetrate through read then use Bao Di Algorithm (providing on WWW bowtie-bio.sourceforge.net/index.shtml) calibrates ALK cDNA order.
Capillary tube order-checking is carried out with 3130xl Genetic Analyser (Applied Biosystems, Inc., Foster City, California), PCR primer and desired cDNAs have identical primer construction or have EA-F-g-S
(5 '-CCACACCTGGGAAAGGACCTAAAG-3 ') and ALK-TK-R2 (5 '-CCTCCAAATACTGACAGCCACAGG-3 ') The sequence of primer.
B. suddenly change EML4-ALK
CDNA is encoded FLAG Ag capture ELISA EML4-ALK variant 1(Soda, M.et al. (2007) Nature 448:561-566) it is inserted into pMX-iresCD8 retroviral vector (Yamashita Y.et al. (2001) J.Biol. Chem.276:39012-39020) the FLAG-labelling EML4-ALK simultaneously expressed in and Mus CD8.Nucleotide change with C1156Y with L1196M is consistent, and the sudden change of ALK introduces plasmid alone or for EML4-ALK (C1156Y),
EML4-ALK (L1196M), or the combination that EML4-ALK (C1156Y/L1196M) expresses.Recombinant retrovirals based on these plasmids Virus uses package cell line, BOSC23 (Pear, W.S.et al. (1993) Proc.Natl.Acad.Sci.USA 90:8392-8396), and use infecting mouse interleukin 3 dependent cells system BA/F3 (Palacious, R.et al. (1985) Cell41:727-734) produce.Its result CD8-positive cell uses miniMACS cell separation post and magnetic Glass dust come purification and with conjugation on the antibody to CD8 (both of which from Miltenyi Biotec, Gladbach, Germany).PF-02341066 is purchased from Selleck.
In order to check the tyrosine phosphorylation of EML4-ALK, BA/F3 cell expressed fusion protein is exposed under ALK inhibitor 15 Hour, EML4-ALK immunoprecipitation from cell lysate goes out to have FLAG(Sigma-Aldrich, St.Louis afterwards, MO) antibody and with to Tyr1604-phosphorylation ALK (Cell Signaling Technology, Danvers, MA) antibody By immunoassay.Vitro kinase measure be at room temperature with synthesis YFF peptide (Operon Biotechnologies, Huntsville, AL) operation 30 minutes, such as previous (Donella-Deana, A.et al. (2005) Biochemistry44:8533-8542) Described.
Example 2-novel mutation ALK contacts with anti-alk tyrosine kinase inhibitor
Patient is the man of 28 years old, does not has smoking history, in April, 2008 clinical stage be diagnosed as adenocarcinoma of lung T4N3M1. In view of tumor does not has any sudden change EGFR to survive, this patient accepts conventional chemotherapy, and its result is that disease continuous several times occurs at brain Shift with in skeleton.In November, 2008, by reverse transcription-pcr sputum analysis and by the fluorescent in situ of tissue samples Hybridization analysis, finds that the EML4-ALK variant 1 of mRNA exists in tumor.Therefore, this patient is registered in PF-02341066 In test, and from the beginning of his physical situation is obviously improved (being reduced to 2 from 4 grades).Although he shows over the course for the treatment of " local response ", his hydrothorax is also non-fully eradicated.But, through the treatment of 5 months, tumor abruptly started to again increase Long, cause hydrothorax increase and form multiple cancer tubercle on double lungs.Patient abandons test in May, 2009, and to thoracic cavity Hydrops carries out Molecular analysis.
Giving ALK inhibitor but tumor is still recovered to increase although lasting, this shows that tumor creates secondary heredity change and has Drug resistance to medicine.Additionally, due to anti-TKIs is often to produce sudden change on kinase target to cause, and to EML4-ALK it Self have passed through may being verified of amino acid whose change.
Respectively the patient tumors treatment expectorant (IDJ-#1) of front and rear and hydrothorax (IDJ-#113) specimen are carried out point Sub-credit analysis understands, and in view of the ratio of the two specimen tumor cell may be different, new-generation sequencing instrument is used for picking up from So cDNAs of the EML4-ALK of specimen has carried out the sequence analysis of the degree of depth.The details result of two specimen is cDNAs and ALK Tyrosine kinase domain identical (Figure 1A), dispersion also carries out Cytosine deaminases by GAII system.As a comparison, EML4 The positive Lines H2228 of-ALK, and other three be also that the positive clinical samples of fused protein carries out phase Same analysis.One known single nucleotide polymorphism rs3795850, is all found to have cDNAs(Figure 1B in four specimen). Additionally, low probability (8.9%) finds at human wild type ALK cDNA(GenBank Accession, NM_004304 in J-#1cDNAs) Nucleotide 4230 position that is equivalent to have the change of a T → C.And, two new changes, send out in J-#113cDNAs There is the change of G → A and C → A nucleotide 4374 and 4493 position that is equivalent to of wild type ALK cDNA now, and its probability divides It not 41.8% and 14.0%.In the kinase domain cDNAs pick up from other specimen, do not occur that other regular changes (go out Existing read rate is >=5%).
The change of these nucleotide uses the order-checking of Mulberry lattice to confirm subsequently.Get rid of this sudden change and be to occur at endogenous wild type ALK In rather than probability in EML4-ALK, detect front end primer target to EML4cDNA with PCR, so that only will melt Mould assembly cDNA expands (Figure 1A).Picking up from T4230C in hundreds of the pattern of fusion cDNAs of J-#1 and do not find change, this shows In original PCR or GAII sequencing steps, it is artifact.
But, the change of G4374A and C4493A can be easily obtained confirmation by the order-checking of Mulberry lattice.For J-#113 Cloning and sequencing 73 merges in cDNA, and 34 clones' (46.6%) of G4374A are positive, 11 clones' (15.1%) of C4493A For the positive, remaining (38.4%) is wild type (Fig. 1 C).Owing to pcr analysis covers two nucleoside in identical product Acid site, does not has product to comprise two sudden changes, and this shows that each sudden change is independent.Around G4374 or C4493 position Genomic fragment expand also by PCR and carry out nucleotide sequencing, its result is that each genome in tumor each changes Be confirmed (Fig. 2).
The replacement result of G4374A and C4493A is in amino acid/11 156 and 1196 position being equivalent to Wild type human ALK On have cysteine → tyrosine and the change of leucine → methionine.
Example 3-novel mutation ALK has the Drug resistance to alk tyrosine kinase inhibitor
Next step research is that ALK inhibitor changes the sensitivity of impact to the aminoacid of EML4-ALK.Wild type EML4-ALK, Single mutation EML4-ALK(C1156Y) and EML4-ALK(L1196M), and double sudden change EML4-ALK(C1156Y/L1196M) Express on BA/F3 cell and the cell after contacting with ALK inhibitor respectively.PF-02341066 is at concentration dependent growths The inhibitory action (Fig. 4 A) of wild type EML4-ALK is expressed on BA/F3 cell.In contrast, cell mutation is expressed or C1156Y L1196M sudden change confirms attenuating notable to drug susceptibility, repeats to test the EML4-ALK showing that BA/F3 cell is expressed (L1196M) than the EML4-ALK(C1156Y expressed) bigger (Fig. 3) to the Drug resistance of PF-02341066.Exist The two sudden change does not cause cell superposition drug-fast to PF-02341066.Therefore, these data show C1156Y It is respectively provided with the Drug resistance to medicine with L1196M sudden change.
By using the immunoassay of ALK phospho-specific antibody on Tyr1604 to verify EML4-ALK tyrosine phosphatase Change.Although BA/F3 cell is exposed under PF-02341066 can substantially suppress wild type EML4-ALK tyrosine phosphorylation, It is not to EML4-ALK(C1156Y) or significant impact (Fig. 4 B) EML4-ALK(L1196M).According to these Existing, kinase assay finding, C1156Y and L1196M of EML4-ALK suddenlys change than wild-type protein PF-02341066 in vitro Suppression sensitivity less (Fig. 4 C) to enzymatic activity.As cytostatic case (Fig. 4 A), C1156Y is compared in L1196M sudden change Sudden change PF-02341066 more difficult to the suppression of kinase activity (Fig. 4 C).
Structure-function relationship between example 4-novel mutation ALK and anti-alk tyrosine kinase inhibitor
Fig. 5 show Cys1156 and Leu1196 based on kinases, ALK kinase domain on the crystal structure of Insulin receptor INSR Position in 3-D solid structure model, the former residuum is positioned at the aminoterminal being connected with prediction spiral α C and namely ties close to ATP- The position of structure tissue medicated cap upper end.Also not about the report of sudden change active on this position in other tyrosine kinase. The Leu1196 of ALK is equivalent to the Thr790 of Thr315 and EGFR of ABL1, and these positions in these kinases are each It is very easy to be mutated into and there is (Deininger, M.et al. (2005) Blood105:2640-2653 of the Drug resistance to TKIs; Linardou,H.et al.(2009)Nat.Rev.Clin.Oncol.6:352-366).These " doorkeeper " positions It is positioned at (Fig. 5) on the bottom surface of ATP-structure organization, and the aminoacid existence in these positions with bulky side chain stops a large amount of Bonding (Shah, N.P.et al. (2002) the Cancer Cell2:117-125 of TKIs;Tsao,M.S.et al.(2005) N.Engl.J.Med.353:133-144).
Therefore, two of the kinase domain at EML4-ALK is had the repellence to multiple ALK inhibitor and has carried out identifying, In view of the two kinds of sudden changes not observing EML4-ALK cDNAs, it is believed that each sudden change is only in the different sub-clones of tumor Vertical generation.
Without bounce-back described above, the cDNAs that the sputum before patient treatment is previously obtained do not contain with C1156Y or The change nucleotide that L1196M sudden change is the same, it is likely to obtain weight in the tumor subcloning procedures treated with PF-02341066 New mutation.But, hydrothorax cannot be checked before the treatment, what patient was admitted to hospital that the initial stage gets rid of the most completely probably already exists Hydrothorax existence may there is C1156Y or L1196M of tumor cell to suddenly change.Under if so, at 5 months Other the most unknown sudden changes may be obtained by tumor in the PF-02341066 treatment phase, and allow its quick growth subsequently. But, the tumor cell sub-clone with C1156Y or L1196M sudden change was difficult to treat at the treatment initial stage, and over the course for the treatment of There is the possibility of amplification.On the contrary, at least 5 months, patient does not has the signal that tumor expands, and this shows C1156Y and L1196M Sudden change occurs during treating with PF-02341066.This viewpoint is supported further by the T790M sudden change practical work of EGFR, It is frequently found gefitinib or erlotinib are had repellence in patient treats with TKIs in advance, and be difficult to find and do not carry out The case (Pao, W.et al. (2005) PLoS Med.2:e73) for the treatment of.
Finding that the doorkeeper position of some tyrosine kinase occurs aminoacid to replace (Kobayashi, S. with in TKIs treatment tumor et al.(2005)N.Engl.J.Med.352:786-792;Pao,W.et al.(2005)PLoS Med.2:e73; Shah,N.P.et al.(2002)Cancer Cell2:117-125;Cools,J.et al.(2003)N.Engl.J. Med.348:1201-1214;Tamborini, E.et al. (2004) Gastroenterology127:294-299).But The report the most not suddenlyd change in this position with EML4-ALK or ALK in advance, various at NPM-ALK, other pattern of fusion ALK's The impact that the artificial aminoacid of doorkeeper position is replaced is verified ((Lu, L.et al. (2009) Biochemistry recently 48:3600-3609).Analyze consistent with interior tumor cell, find that introducing methionine in this position makes NPM-ALK to multiple ALK inhibitor has maximum repellence.
Compare doorkeeper to replace, in other tyrosine kinase also not at α C spiral close to aminoterminal (in ALK Cys1156) report of the active sudden change in this position.With the presence of at the relevant position of EGFR Thr → Ile in NSCLC case The elaboration of change, but drug susceptibility is not verified (Tsao, M.S.et al. (2005) N.Engl.J.Med. 353:133-144).Enzymatic activity allosteric rule for the important α C spiral of serine-threonine kinase is verified
(Hindie, V.et al. (2009) Nat.Chem.Biol.5:758-764).The Cys1156 change directly ginseng of ALK And in the physical action arrived between kinase domain and TKIs.
Equivalent
Those it would be recognized by those skilled in the art that or can determine use but less than conventional test, with the present invention here Described specific embodiment has a lot of equivalent.These equivalents are included in the claim of the present invention.

Claims (18)

1. for determining the chemosensitivity of a cancer patient, thus it is used for the reagent that a kind of ALK inhibitor carries out treating Box, it is characterised in that including: a kind of and one or more sudden change ALK polynucleotide molecules or polypeptide form specific binding examination Agent, the coding C1156Y sudden change of described ALK polynucleotide molecule and/or the ALK polypeptide of L1196M sudden change, described polypeptide bag Include C1156Y sudden change and/or L1196M sudden change；And operation instructions.
2. test kit as claimed in claim 1, it is characterised in that also include a kind of ALK inhibitor.
3. test kit as claimed in claim 1, it is characterised in that described reagent includes one or more nucleic probe, each of which Individual probe includes a polynucleotide sequence, this polynucleotide sequence and a nucleotide sequence complementary, the nucleoside of this complementation Sequences code includes C1156Y sudden change and/or the ALK polypeptide of L1196M sudden change.
4. test kit as claimed in claim 3, it is characterised in that described probe includes a length of 50-107Many nucleoside of individual nucleotide Acid.
5. test kit as claimed in claim 3, it is characterised in that described probe includes oligonucleotide.
6. test kit as claimed in claim 3, it is characterised in that described probe includes cDNA molecule.
7. test kit as claimed in claim 3, it is characterised in that described probe includes RNA molecule.
8. test kit as claimed in claim 3, it is characterised in that described probe includes by the synthetic gene probe of base composition.
9. test kit as claimed in claim 1, it is characterised in that described reagent includes a kind of antibody, a kind of antibody derivatives or Plant antibody fragment, its specific binding ALK polypeptide comprising C1156Y sudden change and/or L1196M sudden change.
10. test kit as claimed in claim 1, wherein said sudden change ALK polypeptide is to include the ALK polypeptide that C1156Y suddenlys change.
11. test kits as claimed in claim 1, wherein said sudden change ALK polypeptide is to include the ALK polypeptide that L1196M suddenlys change.
12. 1 kinds determine a kind of detection compound whether have adjusted one or more comprise C1156Y sudden change and/or L1196M sudden change The method of the activity of ALK polypeptide, it is characterised in that including:
A () contacts with mammalian cell by detection compound, wherein said mammalian cell transfects a structure in advance, makes to turn Dye structure encodes one or more ALK polypeptide comprising C1156Y sudden change and/or L1196M sudden change；And
B () measures this mammalian cell to determine the activity of one or more sudden change ALK polypeptide, wherein relative to a comparison Experiment, detection compound has considerably adjusted activity, and therefore it can be as the actuator of one or more sudden change ALK polypeptide.
13. methods as claimed in claim 12, it is characterised in that the ALK polypeptide of described one or more sudden changes is to comprise The ALK polypeptide of C1156Y sudden change.
14. methods as claimed in claim 12, it is characterised in that the ALK polypeptide of described one or more sudden changes is to comprise The ALK polypeptide of L1196M sudden change.
15. the method as described in claim 12 or 13 or 14, it is characterised in that described control experiment includes mammalian cell table Reach a wild type ALK polypeptide.
16. methods as claimed in claim 15, it is characterised in that the activity of described one or more sudden change ALK polypeptide includes ATP In conjunction with, tyrosine kinase activity, cancer cell proliferation, tumor growth, tumor quantity, apoptosis and neoplasm metastasis.
17. the method as described in claim 12 or 13 or 14, it is characterised in that described control experiment includes that mammalian cell exists Described detection compound is expressed one or more sudden change ALK polypeptide.
18. methods as claimed in claim 17, it is characterised in that described one or more sudden change ALK polypeptide actives include ATP In conjunction with, tyrosine kinase activity, cancer cell proliferation, tumor growth, tumor quantity, apoptosis and neoplasm metastasis.
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